The importance of the problem which makes the object of my study is that despite many years of STD screening programs and important advances in… [307488]

[anonimizat] . Trichomonas vaginalis and Chlamydia are the most commonly diagnosed bacterial STI in many countries and may lead to complications such as pelvic inflammatory disease and infertility. The clinical importance of the T. [anonimizat]. or the T. vaginalis viruses (TVVs; Bar et al., 2015

I [anonimizat], and I am gratefull to the doctors and assistants who helped me during one year time.

.

CHAPTER 1

[anonimizat].

The infection can be transmitted through any type of sexual activity that involves both the genitals and the anus or oral; infection may also be transmitted by contact with blood during sexual activity.

Some diseases are not considered to be formally STDs (eg, hepatitis B or C), but sexual transmission is clearly documented.

[anonimizat] ( formal STD) [anonimizat].

Sexual transmitted diseases according to frequency [source www.cdc.gov]

Bacterial Vaginosis

Chlamydia

Gonorrhea

Viral Hepatitis

GenitalHerpes
Human Papillomavirus (HPV)

Syphilis

Trichomoniasis

Other STDs

HIV & STDs

Complications: pelvic inflammatory disease (PID), [anonimizat]. Some STDs can cause pelvic infections such as pelvic inflammatory disease (PID), [anonimizat]. Abscess, [anonimizat], [anonimizat].

• infection with human papillomavirus (HPV) is a known cause of cervical cancer.

• [anonimizat], during or immediately after birth.

• Since the BTS transmission path is similar, a [anonimizat]. [anonimizat] 50% of cases of gonorrhea are also infected with chlamydia.

A number of individual risk behaviors (such as higher number of sex partners), [anonimizat] (such as higher prevalence of STDs or difficulty accessing health care) contribute to the sexual transmitted disease. [anonimizat], most reported Chlamydia and gonorrhea infections occuring among 15–24-year-. While surveillance data show signs of potential progress in reducing chlamydia and gonorrhea among young people aged 15–24, both the numbers and rates of reported cases of these two diseases continue to be highest in this group compared to other age groups. Both young men and young women are heavily affected by STDs — but young women face the most serious long-term health consequences. It is estimated that undiagnosed STDs only in USA cause 24,000 women to become infertile each year. [.CDC Fact Sheet dec 2014.]

Figure 1.1. Prevalence of STD Source The Lancet

Figure 1.2. Prevalence of most important STD in Europe Source: CDC.gov

CHAPTER 2

TRICHOMONAS VAGINALIS

Parasites are organisms that acquire their nutriments and protective environment through an intimate contact with another living organism, taking all benefits from this relationship. Also, the parasite can cause different degrees of damage to the human host.

Parasites may be classified in simple unicellular organisms – parasitic protozoa or complex multicellular organisms – metazoa – the parasitic helminths or worms.

They may be also classified as endoparasites, the parasites that live within another living organism: intestin, blood and tissues, genital tract and ectoparasites, parasites that live on the external surface of another living organism, on the skin and hair.

A definitive host is the organism in which the adult or sexually mature stage of the parasite lives and in which sexual stages of reproduction occur

The intermediate host is the organism in which the parasite lives during the immature or larval form or in which the parasite undergoes asexual reproduction
The vector is a living carrier (e.g.an arthropod) that transports a parasite from an infected to a non-infected host.

The reservoir is a host (animal, human) that harbors an infection that can be transmitted to humans, even if the animal is a normal host of the parasite.

A carrier is a host harboring the parasite without any clinical symptoms but being capable of shedding the parasite and infecting other people.

Humans may act as:definitive hosts, intermediate host, reservoir host and carrier host.

The Protozoa belong to the kingdom Protista. They are eukariotic unicellular microorganisms. Protozoa inhabit soil and water and relatively few are medically important and cause disease in humas.

Sarcodina are typically amoeboid and can move due to pseudopodia or protoplasmic flow. They are represented in humans by Entamoeba, Naegleria and Acantharnoeba.

Mastigophora, the flagellates have one or more flagella and in some cases an undulating membrane.

Mastigophora include intestinal flagellate Giardia and urogenital flagellate Trichomonas; blood and tissue flagellates like Tripanosoma and Leishmania.

Sporozoa has a complex life cycle including sexual and asexual reproductive phases, usually involving 2 different hosts. They do not have mobility, or organelles of motion. The subclass Coccidia contains the human parasites Isosopora, Toxoplasma and Cryptosporidium. Among the Haemosporina (blood sporozoa) is Plasmodium species. Pneumocystis has recently been shown to be a member of fungi rather than the Protozoa.

Ciliophora is a complex protozoa bearing cilia distributed in rows and patches with two kinds of nuclei in each individual. The only human parasite from this group is represented by Balantidium coli.

Microsporidia includes intracellular parasites, infecting many hosts, but in humas are opportunistic parasites of immunocompromised hosts. They were formerly classified as sporozoa because of polar filaments inside a spore.

Trichomonas vaginalis is a urogenital flagellate protozoan and is the main species of the genus that produces human disease.

Other species are Trichomonas hominis and Trichomonas tenax, that colonise the intestinal tract and mouth. Trichomonas hominis i It is thought to be non-pathogenic although is often identified in human diarrheic stools. Because of the fecal-oral transmission rout, T. hominis is more frequently reported in children than in adults.

Trichomonas vaginalis is the etiologic agent of a sexually transmitted disease called trichomoniasis.

MORPHOLOGY

Trichomonas vaginalis exist only as trophozoite and do not produce cysts. The trophozoite is a pear-shaped (ovoid) organism, measuring 10 µm in diameter and 20 µm in length. It has a central skeletal rod or axostyle, an anterior nucleus, 4 anterior flagella and a recurrent flagellum attached to the pellicle to form an undulating membrane. Due to flagella, Trichomonas vaginalis is extremely motile, with darting motions, the motility contributing to its pathogenicity. Mobile trichomonas can be seen in wet mounts

Figure 2.1. Trichomonas vaginalis morphology. Source: www.phsource.us

It reproduces through binary fission .

Because there are not resistance forms,transmission is always interhuman directly (Fig)

Figure 2.2. Trichomonas division and transmission. Source CDC.gov

EPIDEMIOLOGY

Trichomoniasis is a common sexually transmitted disease disease (STD). About 25-50% of sexually active women are infected during their life. Trichomonas vaginalis is typically transmitted through vaginal, oral, or anal sex with an infected individual. Asimptomatic people ( about About 70% of them) can transmit the infection as well. It is possible, but very rarely that trichomoniasis is aquired from soiled towels, baths, jacuzzis andmedical instruments. It can also be passed from an infected mother to the baby during delivery.

Recent studies showd that TV is more prevalent that Chlamydia trachomatis, Neisseria gonorrhoeae, and syphilis combined. The global prevalence of TV has been estimated at 8.1 % for women and 1.0 % for men [1, 13]. These rates may be underestimates as they are derived from studies that used microscopy rather than the more sensitive nucleic acid amplification tests .

PATHOGENICITY, PATHOLOGY

Women are generally symptomatic having a vaginitis with a watery, foul-smelling vaginal discharge, itching, burning, dysuria, dyspareunia. The typically symptomatology get worse during menses and pregnancy.

The gynaecological examination reveals a reddened friable endocervix (without therapy, in time this lesions can lead to malignancy).

Figure 2.3. Vaginal discharge in Trichomonas infection

Men are usually asymptomatic (urine washes the urethra).

When they are symptomatic (10%) they have a urethritis with a watery discharge, itching, burning, disuria or mild prostatitis.

In addition to causing symptomatic vaginitis, infections by T. Vaginalis are increasingly recognized as being associated with other serious clinical outcomes, including premature delivery, low birth weight, cervical cancer, and prostate cancer, as well as an increased risk of infection by HIV and human papillomavirus.

Recent studies show that to establish and maintain infection the parasite Trichomonas vaginalis adheres to host cells, a process that is poorly understood T. vaginalissecretes small vesicles called exosomes that are capable of fusing with and delivering their contents to host cells. [12] Parasite exosomes were found to induce changes in the host cell and to mediate the interaction of T. vaginalis with host by increasing the adherence of the parasite to host cells. Exosomes have been primarily studied in mammalian cells where they have been shown to mediate intercellular communication and have been implicated in processes including development, antigen presentation and cancer metastasis. The data can extend the function of exosomes to mediating host∶parasite interactions, cellular communication between two species and promoting colonization of an extracellular parasite. Research on T. vaginalis exosomes holds the potential for developing applications that would allow exosomes to be used in detecting and diagnosing trichomoniasis and for targeting drugs to the site of infection.

LABORATORY DIAGNOSIS

Diagnosing a trichomoniasis usually requires a sample of discharge, during a pelvic exam for women and a urethral swab or urine sample for men. But  the least invasive way to test for trichomoniasis in men or women is simply via a urine sample. The  first catch or first void urine sample– meaning that the urine sample is the first 20-30 milliliters of urine in the initial stream of urine. Individuals should not have urinated for at least one hour prior to specimen collection.

The most common testing method for women is a “wet mount” or “wet preparation,” which allows for evaluation of the discharge under a microscope to confirm the presence of the parasite. A Trichomonas vaginalis culture can also be used to confirm the infection after it is discovered during a woman’s Pap smear if the presence of the parasite can’t be confirmed using microscopy.

Microscopic examination of wet mounts from vaginal or urethral discharge show the highly motile parasites with darting motions.

The sensitivity of this test varies from 38% to 82% and is dependent on the inoculum size because fewer than 104 organisms/mL will not be seen. As well, the need for the specimen to remain moist and the experience of the observer are important variables. The size of the trichomonad is approximately the same as that of a lymphocyte (10 µm to 20 µm) or a small neutrophil; when not motile, a trichomonad can be difficult to differentiate from the nucleus of a vaginal epithelial cell. Motility is very dependent on the temperature of the specimen. At room temperature in phosphate-buffered saline, the organism will remain alive for more than 6 h; however, the motility of the organisms becomes significantly attenuated. This wet mount examination is clearly the most cost-effective diagnostic test, but the lack of sensitivity contributes to the underdiagnosis of the disease. Because viable organisms are required, delay in transport and evaporation of moisture from the specimen reduces motility and, consequently, diagnostic sensitivity [1].

The same appearence may be in urine sediment microscopic examination, during urine test.

Vaginal and cervical smears, stained Giemsa or Papanicolaou

Acridine orange staining ( Figure )

Fig. 2.4. T.vaginalis stained Giemsa Source:www. JCDR

Other tests:

Fluorescent antibody assay (staining)

Polymerase Chain Reaction (PCR) for nucleic acid detection

Recombinant DNA technology has been adapted over the past decade as a diagnostic tool. A variety of primers have been tested, including primers initially reported by Riley et al (17). This has subsequently been commercialized into the Affirm VP system (Becton, Dickinson and Company, USA). Depending on the studies performed, the highest sensitivity of this technique is less than 90%, and false positives have been reported post-treatment because of DNA from dead organisms (18). A recent article by Crucitti et al (19) indicated that in an African population, some primers were more effective than others when compared with culture-based techniques. Other studies have shown a higher sensitivity rate, which also appears to be dependent on which specimens are being used for testing. In general, urine sampling for T vaginalis in women is less effective than supervised vaginal swabs, and self-administered vaginal swabs have had variable results depending on the population tested (20). Specificity with this method can be less than with wet mount due to false-positive tests.

Dot blot hybridization has also been used, employing a 2.3 kb T vaginalis DNA fragment as a probe. Unfortunately, this probe has been shown to be unstable as a radioactive probe but can be improved with a fluorescent-labelled technique (21). Its role in determining asymptomatic carriers has not been established. Nucleic acid detection of T vaginalis is not generally available (only in reference laboratories).

Identification of T vaginalis in men

Because the majority of men are asymptomatic carriers, the diagnosis of T vaginalis is usually not made, and the male partner is identified and treated with metronidazole at the same time that the female partner is treated. In the small number of males who are symptomatic, urethral discharge collected with a swab would provide the best results using the broth culture technique, with sampling ideally done before first-voided morning urine. Using the urine-based polymerase chain reaction (PCR)-ELISA technique described by Kaydos-Daniels et al, it has been shown that in a Malawian population, PCR on first-catch urine had a sensitivity of 92.7% and a specificity of 88.6%.

Figure.2.5.Microscopical images of Trichomonas vaginalis infection. Source www.haemosexual.com

Figure 2.6. T. vaginalis . Source http://medstat.med.utah.edu/parasitology/tvagim.html

Figure 2.7. T. vaginalis and polymorph leukocytes. Source: fertilab.net

In difficult cases can be used cultivation on special medium (Lowe's, a nutrient broth plus horse serum and antibiotics).

Broth culture technique has been the gold standard for T vaginalis for more than 40 years. [ 2] The inoculum size required is only in the range of 102 organisms/mL and the growth of the organism is easy to interpret. The standard broth is Diamond's TYI medium in glass tubes . Incubation periods ranging from two to seven days are required to identify T vaginalis in culture. Contamination with bacteria is a major problem, even with broth cultures spiked with antibiotics to eliminate vaginal flora. Passage of the culture after two to three days to reduce the bacterial contamination may be required to definitively identify the T vaginalis culture. The organism has the capacity to enter lag growth and, even in well-established axenic culture, can sometimes have attenuated growth for 24 h to 48 h before re-establishing its characteristic log/day growth. Because of the expense, culture techniques have not been readily available but would be the most effective way of establishing the true epidemiology of T vaginalis, particularly where STI clinics are remote from the clinical laboratory. To circumvent these problems, the InPouch system (BioMed Diagnostics, USA) and similar culture systems have been developed whereby the specimen is put into a two-chambered bag, allowing sampling for immediate wet mount microscopy and incubation for culture (14). In some studies, this has been shown to be at least as effective as Diamond's medium in glass vials. T vaginalis is an anaerobic organism that grows more slowly under aerobic conditions. Thus, CO2 incubation has been recommended for optimal recovery.

Fig. 2.8. InPouch system (BioMed Diagnostics, USA. BioMed’s InPouch™ TV test is a microbiology sample collection, transport, and culture device that allows for simultaneous growth and observation of Trichomonas vaginalis, the parasite responsible for the sexually transmitted infection trichomoniasis

Cultivation on cell cultures is more sensitive, enabling the observation of T vaginalis from an inoculum containing as few as 3 organisms/mL. However, cell culture is expensive, inconvenient and even more prone to vaginal bacterial contamination. This technique requires pretreatment of the specimen with antibiotics using Diamond's TYI medium as a transfer medium, followed by subsequent passage onto the cell cultures. A combination of TYI and cell culture medium (2:1) supports both the monolayer and T vaginalis growth. Despite its high sensitivity, this method has not been used outside a limited number of studies

Antigen detection in specimens in also available ( figure..)

Fig. 2.9. Antigen detection kit

Quality control and proficiency testing

Examination for T vaginalis is most often done as part of the investigation of vaginal discharge. It is recommended that the examination is included as part of any routine examination for vaginitis. pH evaluation with pH paper can help to rapidly differentiate T vaginalis from yeast at the bedside. The vaginal pH, which is normally pH 4.5, is not altered by yeast infection but is elevated in bacterial vaginosis and often rises above pH 6 in florid trichomoniasis. Quality concerns include the need to have the microscopic examination done immediately after specimen collection; even then, keeping the sample moistened with saline or phosphate-buffered saline is key. Alternatively, the specimen can be collected into an InPouch kit or into Diamond's medium to ensure survival of the organism during transportation to the laboratory. Where specimen transportation is suboptimal, the report of a negative finding should include a reference to the possibility of a false-negative result. Stained preparations can be difficult to read; therefore, the laboratory must take the steps necessary to ensure the ongoing competence of its microscopists. Cultures and positive specimens can be used to familiarize staff with the characteristic movement of motile T vaginalis. The laboratory should maintain communication with its clinicians to ensure that specimens submitted for the diagnosis of trichomoniasis are vaginal and not cervical [2].

CHAPTER 3

TREATMENT AND PREVENTION OF T.VAGINALIS INFECTION

For the therapy may be indicated Metronidazole (Flagyl), Tinidazole (Fasigyn) orally in single dose or twice a day for 7 days. Treating all sexual partners is compulsory to avoid re-infection.

Resistance to metronidazole, the first-line treatment for trichomoniasis, is on rise.

Metronidazole should not be used during the first semester of pregnancy because of it potential teratogenic effect.

Prevention is made by safe sex practices, including the use of condoms. It is also important to stop any sexual activity of infected persons during the treatment .

Treatment with imidazoles

For nearly four decades, metronidazole (MTZ) has been the treatment of choice for Trichomonas vaginalis infections [3]. MTZ belongs to the 5-nitroimidazole drug family and is reported to have about a 95 % success rate in curing TV along with its related compounds such as tinidazole (TNZ) and seconidazole [4]. The World Health Organization (WHO) guidelines for treatment of TV include: MTZ or TNZ 2 gm single dose as the recommended regimens, and MTZ 400–500 mg BID 7 day dose as the alternative treatment regimen. Abstinence from alcohol use should continue for 24 h after completion of MTZ or 72 h after completion of TNZ. If a patient fails single dose MTZ therapy they can be given single dose TNZ or 7 day dose MTZ. If this fails, 2 g MTZ or TNZ for 5 days can be administered. If this fails and there is no history of sexual re-exposure, a consultation for medication resistance testing should be done. http://www.cdc.gov/std).

Treatment among pregnant and lactation women. MTZ is a class B drug and several meta-analyses have found it to be safe in pregnant women in all stages of pregnancy [5]. TNZ has not been evaluated in pregnant women and remains a class C drug. Treatment with 2 g MTZ is recommended by CDC at any time during pregnancy [6] whereas WHO does not recommend treatment in the first trimester unless it is indicated for prevention of untoward birth outcomes. Both entities suggest 2 g dose.

In lactating women who are administered MTZ, withholding breastfeeding during treatment and for 12–24 h after the last dose will reduce the exposure of the infant to metronidazole. For women treated with TNZ, interruption of breastfeeding is recommended during treatment and for 3 days after the last dose.

Treatment of persistent TV or allergies to MTZ/NTZ. Persistent TV is usually treated with multi-dose MTZ or TNZ. The most common reactions reported from metronidazole are urticaria and facial edema, while other adverse reactions include flushing, fever and anaphylactic shock from immediate-type hypersensitivity have been reported. De-sensitization can be done, but only has about a 42 % cure rate. If TV remains persistent or the patient is allergic to these medications, other intravaginal treatments have been studied or are under investigation TV including: Acetarsol , Boric acid, Furazolidone , and Paromomycin [7,8]. Nitrazoxanide was examined as an alternative oral agents for MTZ-resistant TV but was not found to be very effective. Several combination therapies including TNZ plus ampicillin and multi-dose NTZ [81]. Some plant extracts have shown anti-TV activity, but these have not yet been tested in clinical trials .

Treatment among HIV-infected women. In an randomized clinical trial among HIV-infected women with TV, multi-dose MTZ was found to be superior to single dose treatment. Further analysis revealed that the superiority is only in the presence of bacterial vaginosis (BV) [9].

It has been estimaged that if CDC recommendation for TV screening and treatment among HIV+ women is followed, that the lifetime cost of new HIV infections prevented would approximate US $159,264,000 via new HIV cases of secondary to female-to-male transmissions prevented [10].

Repeat/ persistent infections. Repeat infections are common, ranging from 5–31 % , and share similar sequelae to primary infections[11]. While it is clear that the TV repeat infection rate is unacceptably high, the source of these repeat infections is less clear. Possible sources of retest positives after treatment are: re-infection from an untreated/infected baseline partner, infection from a new partner, or treatment failure. Each of these sources of retest positives requires a different approach to prevent ongoing infection . If the cause is re-infection, then assuring the original partners are treated is needed. If the source is a new partner or treatment failure, then rescreening is needed.

CHAPTER 4

MYCOPLASMA AND UREAPLASMA

Mycoplasma

The genus Mycoplasma is part of the Mollicutes class (with soft membrane). Prokaryotic organisms of very small size: 150-250 nm, have a small genome but contain both DNA and RNA, a deformable cell membrane that contains sterols and are completely devoid of cell wall. (Figure )

Figure 4.1. Mycoplasmsa ( E.M. ) source: K.Toddar

The most important species for human pathology are[14].:

Mycoplasma pneumoniae, which causes respiratory infections

Mycoplasma hominis, Mycoplasma genitalium and Ureaplasma urealyticum, which cause genital infections.

M. pneumoniae is aerobic, while the other species are aerobically optional anaerobes. Gram stains gram stain (Gram negative).

They grow in vitro on acellular culture media, but they are pretentious bacteria. Forms characteristic colonies with the appearance of egg mesh.

These bacteria are very stubborn and resistant to most antibiotics used for treatment.

Pathophysiology.

Mycoplasms attach to the surface of epithelial cells at the level of some red blood cells I-like receptors. Humoral immune response leads to the appearance of anti-I or cold agglutinin antibodies, which act as autoantibodies. After attachment, mycoplasms produce a direct cytotoxic effect due to the release of hydrogen peroxide, or lysis of the epithelial cell as a result of the inflammatory response and humoral immune response.

The possibility of systemic infections is reduced.

Transmission and clinical manifestations.

M. pneumoniae is transmitted by aerogenic route via Pflugge drops, with infections being more common in the fall and winter. Most cases are upper respiratory tract infections and only in 5-10% of cases the infection progresses to tracheobronchitis or pneumonia, usually autolimitate. Pleural damage is possible.

In general, symptomatology is unspecific, onset is insidious, with subfebrile state, headache. Persistent non-productive cough is the most characteristic manifestation. The pharyngeal mucosa is erythematous, but laterocervical adenopathy may be absent. Another feature of Mycoplasma pneumonia is the discrepancy between few clinical signs and obvious radiological appearance of pneumonia ( Figure)

Figure 4.2. Typical radiological appearance of pneumonia with Mycoplasma pneumoniae

There may also be extra-respiratory manifestations: cutaneous, cardiac, neurological, autoimmune hemolytic anemia.

In people with sickle cell disease or other hemoglobinopathies, Mycoplasma pneumonia evolves more severely, with significant pleural effusions and respiratory failure.

Mycoplasma hominis, Mycoplasma genitalium and Ureaplasma urealyticum are sexually transmitted.

From recent survey carried out, it has been discovered that up to thirty to fifty percent of women and men that are sexually active are infected by Mycoplasma hominis. Another bacteria that infect over fifty percent of women that are sexually active and five to twenty percent of men that are sexually active is the Ureaplasma urealyticum.

These bacteria have colonized about one-quarter of new born babies, but it is expected that after three months from birth, the colonization rates will drop drastically. Premature babies are more susceptible to this colonization (almost half of the whole premature babies less than thirty-four weeks’ of gestation period may be infected with the bacteria at birth if their mothers are infected by the bacteria. Mycoplasma hominis infection can be spread when there is direct contact with the host, particularly during all types of sexual activities. The bacteria can also be spread by mothers during the gestation period and delivery.

The infection is caused by bacteria called Mycoplasma hominis can be spread through sexual intercourse (either through vagina, oral, or anus), and directly from mother to infant by colonization or nosocomial transplant of tissues. The black race are more infected by Mycoplasma hominis than any other races – the reason still unknown, hence it cannot be concluded that it is a racial or socioeconomic condition.

In most cases, people with Mycoplasma hominis infection do not have symptoms. If there are any, then it will definitely be difficulty and pain when urinating and discharge of fluid from urethra. Joint pain and respiratory problems may also be signs of this bacterial infection common with individuals with fragile immune system, but this is a very rear scenario.

Although vaginal discharge is not caused by genital mycoplasma, urethritis (a disease caused by mycoplasma bacteria) may likely contribute to vaginal infection which may lead to discharge of fluid. There may also be signs of pelvic pain as a result of pelvic (inflammatory) disease caused by mycoplasma or other STDs. In new born babies, it can be very subtle. Changes in heart rate and blood pressure, uneasy breathing and fever are the first symptoms of most problems.

Laboratory Diagnosis Tests :

Mycoplasma only grows in acellular cultures in the presence of various growth factors and at a constant temperature of 35 to 37 °C.

Fifure 4.3. Comparison between 3 different methods for Mycoplasma culture, identification and titration o clinical specimens and various dilutions of U. parvum ATCC 27815 (Up) and M. hominis ATCC 27618 (Mh) strains [J.M. Rousee, C. Rieder-Monsch, T. Gueudet]

Method 1 : collection by a Rayon swab, transfer into the suspension medium of the Mycoplasma Duo kit (Bio-Rad France) and microplate seeding according to manufacturer's instructions

Method 2 : collection and transport by Eswab™ (COPAN) and transfer of 200 μl of ESwab™ medium into the suspension medium of the Mycoplasma Duo kit before microplate seeding

• Method 3 : collection by ESwab™ and direct seeding of the microplate by 100μl of Eswab™ medium

Transport < 48 hours at room temperature

Inoculation of an A7 agar (BioMérieux, France) as gold standard, in each case

Incubation, reading and interpretation according to manufacturer's instructions

Source RichDavis PhD

Figure 4.5. Mycoplasma Identification and Susceptibility Test Kit (MIESTK1)

Creative Diagnistic

Figure 4.6. Biochemical tests

The most reliable test for Mycoplasma hominis infection is PCR test. This detection is specific, sensitive, and offers a same-day outcome.

Although regular plate cultures can be occasionally used to detect this organism, it is a non-sensitive and very slow approach to detect the presence of the bacteria.

Treatment

In upper respiratory tract infections, antibiotic treatment is not necessary.

In pneumonia, although it is self-limited, antibiotic therapy is recommended to reduce the duration of the disease and reduce the risk of contact with the patient.

– Antibiotics of choice are macrolides (Erythromycin 500 mg / 6h / PO 7 days, Clarithromycin 500 mg / 12h / PO 7 days, Azithromycin 500 mg / day / PO 1 day, then 250 mg / day 4 days, adult doses). adults, children, and even pregnant women, after assessing the benefit-risk ratio.

– Tetracyclines (Tetracycline 500 mg / 6h / PO 7 days, Doxicillin 100 mg / 12h / PO in adults) may be given to adults and children over 8 years but are contraindicated in pregnant women. – fluoroquinolones (Levofloxacin is recommended at -500 mg / day / PO, but also Ciprofloxacin, Ofloxacin), only in adults with community-acquired pneumonia; are contraindicated in children and pregnant. In genital infections Tetracycline and Doxycycline are choice antibiotics, and they also have a good effect on Chlamydia trachomatis. However, macrolides are recommended for pregnant women.

Resistance to antibiotics

Often named the “naked bacteria”, because lacking cell wall, M. genitalium is not susceptible to antibiotics that inhibit cell wall formation. Besides the limited range of antibiotics available for managing this organism, the lack of easy and routine methods to determine antimicrobial susceptibility further adds to the problem. However, the inhibition of M. genitalium growth in Vero cell cultures provides an opportunity to determine antimicrobial susceptibility by comparing the proportion of DNA level of M. genitalium controls growing under identical culture conditions.[ Hamasuna R et al, 15]  Tetracyclines, especially doxycycline are still used widely for therapy, but gradually their cure rates have decreased and resistant isolates were identified. Macrolides, especially azithromycin were the second group to be used extensively for managing M. genitalium infections, but again the regimens used were a matter of controversy with present guidelines discouraging the use of a single 1 g dose regimen. Quinolones were initially active against macrolide-resistant organisms but reports of resistance to moxifloxacin are available in recent times. Newer drugs like josamycin and pristinamycin are being used nowadays for the treatment of multidrug-resistant (MDR) organisms. [16, 17]

Tetracyclines

Doxycycline was the most extensively used drug among this group but showed poor response rates in the range of 30%–40%.  Ironically, in vitro data from one study showed that minimum inhibitory concentration (MIC)50 and MIC90 of doxycycline for M. genitalium were 0.25 and 1 mg/L, respectively, contradictory to the clinical efficiency. To date, neither any specific mutations nor the exact explanation for this discrepancy has been reported. [18]

Macrolides

The prominently used macrolide for M. genitalium infection is azithromycin. Traditionally, 2 regimens of azithromycin have been used. Single-dose regimen of 1 g and extended-dose regimen of 1.5 g were given over a period of 5 days. The latter regimen had shown efficacy in Mycoplasma pneumoniae infection, and hence it was predicted that it would be more effective against the slowly growing M. genitalium too; so, it was adopted in many European countries. [19]  The cure rate with the single-dose regimen was initially 85% but this gradually declined as per the studies from various geographic regions. A study by Manhart et al showed only a 40% cure rate in subjects compared with 30% cure rate in those receiving doxycycline.Similarly, another study from Melbourne (Australia) by Twin et al showed a decrease in cure rate from 84% (2005–2007) to 69% (2007–2009). In Greenland, where chlamydial infections are common and a single dose of azithromycin is used for its treatment, almost all M. genitalium infections are caused by macrolide-resistant strains. [20].  Recent guidelines discourage use of the single 1 g dose regimen in light of the emerging macrolide resistance associated with this regimen.[21] A study by Anagrius et al showed that none of the 77 patients treated with the extended regimen developed macrolide resistance. In another study, comparing both regimens in macrolide-susceptible strains showed new onset resistance in 6.5% of subjects receiving the extended regimen and 10% in those receiving the single-dose regimen.The probable cause for the failure of the single-dose regimen may be related to the load of organisms in the affected individuals as shown in a study by Bissessor et al where an increase in the organism load by every log10 was associated with chances of failure of this regimen. Moreover, injudicious use of this regimen without confirming the eradication of M. genitalium might have given rise to spontaneous mutations in the surviving organisms. Ever since the first report of macrolide resistance in 2006, there has been a rise in the resistance rate. As per a recent study among 946 subjects from different geographic regions of USA, the rate of macrolide-resistant M. genitalium infection was 50.8% in females and 42% in male subjects. The mechanism of macrolide resistance involves an alteration in the ribosomal proteins that prevent the binding of the drug to the ribosomes. Mutations have been identified in V region of the 23S rRNA and the L4 and L22 ribosomal components. The predominant mutations identified in the 23S rRNA gene are A2058G, A2059G and A2058T; with the latter being the most common. The unreasonable use of azithromycin for respiratory tract infections and single copy number of the gene are likely responsible for rising macrolide resistance. Thus, newer macrolides like josamycin have been tried for the management of azithromycin-resistant M. genitalium infection. However, mutations associated with resistance in josamycin have also been reported recently. It has been noticed that A2058G and A2059G mutations lead to resistance to the 15-membered macrolides such as azithromycin. Also, it has been found that the same A2059G mutation leads to high-level resistance to the 16-membered macrolides too, such as josamycin. In addition to this, mutation at position A2062 of the 23S rRNA gene can lead to high-level resistance to josamycin (16-membered macrolide) but surprisingly, not to azithromycin (15-membered macrolide), suggesting a difference in the binding site. However, to date, it is unknown whether A2058G/A2059G and A2062G mutation can co-occur in the 23S rRNA gene.

Fluoroquinolones

Moxifloxacin, a fourth-generation fluoroquinolone, has been the most frequently used second-line drug. Its use for management of these infections was reported for the first time in 2006 and many initial studies had shown a cure rate approaching 100%. [ But, recent reports document treatment failures with moxifloxacin, especially in the Asia-Pacific region, with many subjects having an infection with strains resistant to both macrolides and fluoroquinolones. Another study from Japan showed an increasing rate of fluoroquinolone resistance among M. genitalium isolates, with a rise from 20% in 2011 to 47% in 2013.

The first report of a mutation associated with moxifloxacin resistance in M. genitalium was from Sydney, Australia. Mutations in the DNA gyrase genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) are associated with resistance. A study from Japan, identified mutations in the quinolone resistance determining regions of the parC gene as the cause of resistance in moxifloxacin and other fourth-generation quinolones.The mutations in the positions Ser83 and Asp87 (MG numbering) are found in the resistant isolates. The moxifloxacin resistance rate varies in different parts of the world; a rising trend (47%) has been noted in Japan while a lower incidence of 5% in London (UK) and 15% in an Australian STI clinic has been reported. [22]. Despite the reported resistance mutation, no correlation has been established between the rising MIC values and treatment failure rates.

Detection of antimicrobial resistance (AMR)

All strains of M. genitalium isolated from clinical samples before 2003 were susceptible to macrolides but since then the number of resistant strains has increased. With this in mind, all samples with a positive result in NAAT for M. genitalium should ideally be tested for macrolide resistance mutations. With the increasing macrolide resistance, there is a need for an assay to diagnose Mycoplasma and detect macrolide resistance simultaneously. Recently, a multiplex assay named MG 23S assay was developed that employs novel PlexZyme™ and PlexPrime™ technology to diagnose M. genitalium infection and detect 5 mutations involved in macrolide resistance. [23]

Figure 4.7. ResistancePlus MG* is a multiplex qPCR test for detection of Mycoplasma genitalium and five macrolide resistance markers from male and female urine and swab specimens. Proprietary PlexZyme® and PlexPrime® technologies improve multiplex performance compared with other probe-based tests allowing for multiple mutation detection in a single well. Source: www.plexpcr.com

Figure 4.8. Plex prime principle. Source: www.plexpcr.com

A total of 400 samples were evaluated with this assay and the results were compared with the reference quantitative PCR method with high-resolution melt analysis. The sensitivity for M. genitalium diagnosis and mutation detection was shown to be 99.1% and 97.4%, respectively, and the specificity for the same was 98.5% and 100%, respectively.[23] Use of such assays, should be helpful in choosing the appropriate antibiotics for managing the infection. Further data and research will decide the possible future use of this kit for diagnosis. Mutations mediating resistance to moxifloxacin can also be detected by molecular methods that are based on parC gene sequencing. [However, there does not exist a fine correlation between various mutations in parC and in vitro moxifloxacin resistance.To date, no commercial assay has been approved by FDA due to the lack of validation of these developed platforms. Recently, an automated Aptima platform targeting the 16S rRNA is under comprehensive validation and may generate superior results.

Management of M. genitalium infection

Patients with M. genitalium infection are advised to maintain abstinence from unprotected intercourse until both sexual partners have completed the treatment and are symptom-free. Both sexual partners should be screened for other STIs and informed about the risk of transmission and imminent complications. In cases where a partner does not get tested, the same treatment is to be offered as given to the index patient. A test of cure should also be performed routinely for all patients in view of the increasing prevalence of macrolide resistance, which may exist prior to initiation of therapy or can evolve during therapy with a macrolide. Furthermore, M. genitalium infection during pregnancy can jeopardize the health of the fetus as well as the mother, especially in terms of susceptibility to preterm labor and spontaneous abortions. The problem is further aggravated by the absence of safe options for treatment of infection caused by macrolide-resistant strains during pregnancy, hence treatment of such infections is often withheld till completion of pregnancy. Pristinamycin, due to its safety profile, has proven to be a ray of hope for treatment of such resistant infections during pregnancy. The neonates of infected patients should be observed for development of conjunctivitis and respiratory tract infections.

Current treatment options

In view of the increasing resistance among M. genitalium for macrolides and quinolones, the treatment regimen should be short and convenient to the patient in order to ensure adherence. The most recent European guidelines – 2016 have divided the treatment depending on whether the infection is complicated or not and presence/absence of macrolide resistance among the isolates. Similarly, another European guideline for the management of NGU in 2016 has also given a stepwise approach for treatment. [25] Macrolides are still recommended as the first-line antibiotics for M. genitalium infections. Newer antibiotics like josamycin and pristinamycin have also been included in the guidelines.

Doxycycline has a low cure rate of 30-40%, but does not increase resistance. Azithromycin has a cure rate of 85-95% in macrolide susceptible infections. An extended course appears to have a higher cure rate. An increasing prevalence of macrolide resistance, most likely due to widespread use of azithromycin 1 g single dose without test of cure, is drastically decreasing the cure rate. Moxifloxacin can be used as second-line therapy, but resistance is increasing. Uncomplicated M. genitalium infection should be treated with azithromycin 500 mg on day one, then 250 mg on days 2-5 (oral), or josamycin 500 mg three times daily for 10 days (oral). Second line treatment and treatment for uncomplicated macrolide resistant M. genitalium infection is moxifloxacin 400 mg od for 7-10 days (oral). For third line treatment of persistent M. genitalium infection after azithromycin and moxifloxacin doxycycline 100 mg two times daily for 14 days can be tried and may cure 30%. Pristinamycin 1 g four times daily for 10 days (oral) has a cure rate of app. 90%. Complicated M. genitalium infection (PID, epididymitis) is treated with moxifloxacin 400 mg od for 14 days. [2016 European guideline on Mycoplasma genitalium infections].

The symbiosis of Trichomonas vaginalis and Mycoplasma hominis is the first described association between two obligate human parasites. Trichomonas is the niche and the vector for the transmission of M. hominis infection. This clinically significant symbiosis may affect T. vaginalis virulence and susceptibility to treatment. The aims of this study were to investigate the intracellularly present Mycoplasma and Ureaplasma species in T. vaginalisstrains isolated from the vaginal discharge of infected women as well as to trace the diversity pattern among the species detected in the isolated strains.

The clinical importance of the T. vaginalis infections has been reportedly associated with dysbiosis of the urogenital tract microbiome and with the co-existence of the parasite with opportunistic microorganisms including the intracellularly hosted Mycoplasma spp. (Bar et al., 2015; Margarita et al., 2016). The Trichomonas viruses constitute the first double-stranded RNA (dsRNA) viruses associated with the parasite, and the notion that the trichomonads infected by the TVVs may present prolonged survival within the human host due to immunogenic proteins expression modulation is not new (Goodman et al., 2011).

The simultaneous presence of protozoan and the bacteria or the viruses exerts a modulatory effect on innate immunity and the inflammatory process (Rappelli et al., 2001; Hirt and Sherrard, 2015). T. vaginalis and M. hominis are known to have the same arginine dihydrolase (ADH) pathway, which serves as an alternative source of ATP through arginine scavenging. Subsequent arginine depletion blocks nitric oxide (NO) production by host macrophages, alters host defense mechanism and ameliorates the parasite survival (Morada et al., 2010). Furthermore, the extensively studied symbiotic pattern of M. hominis hosted by T. vaginalis is proved to play a key role in inflammation during trichomoniasis, thus enhancing the severity of the disease (Dessi et al., 2005, 2006; Vancini and Benchimol, 2008; Vancini et al., 2008; Morada et al., 2010; Fiori et al., 2013). T. vaginalis hosting M. hominis have also been found to bear higher phagocytic activity than Mycoplasma–free isolates (Cirillo et al., 1997).

The concept of reductive evolution of the mycoplasmal genome from Gram-positive bacteria, based on comparative genomics, justifies both structural and functional alterations (Razin et al., 1998). Mycoplasma species, belonging in the class of Mollicutes, have developed a small genome which lacks biosynthetic pathways involved in macromolecule building blocks synthesis, therefore explaining the absence of cell wall (Liu et al., 2012). The Mycoplasmagenome also carries a minimal set of energy metabolic genes with a restricted adenosine triphosphate (ATP) supply which is essential for their parasitic mode of life (Razin, 1997). Despite the limited genetic redundancy, Mycoplasma spp. present a rather interesting mechanism of self-protection based on their ability to modulate the inflammatory response of the host immune system (Razin et al., 1998).

Among Mycoplasma spp., M. hominis is the most frequently isolated microorganism from the genital tract of both males and females, acting either as part of normal flora or as a putative pathogen responsible for a variety of urogenital infections (Ladefoged, 2000). The Mollicutes represent the most common pathogens associated with T. vaginalis infection with Mycoplasma hominis and Candidatus Mycoplasma girerdii as the predominant strains isolated in trichomoniasis specimens (Koch et al., 1997; Fettweis et al., 2014). The symbiotic phenomenon of M. hominis hosted by the pathogenic protist has received special interest though little is known about the intracellular colonization of T. vaginalis by other Mycoplasma spp. (Martin et al., 2013). The study of the symbiotic relationship between T. vaginalis and M. hominisprovides information about (i) the association between two obligate human pathogens and (ii) an additional role of T. vaginalis as a novel niche for M. hominis. Infected T. vaginalis isolates act as M. hominis carriers mediating transmission in other protozoa and human-derived epithelial cells (Rappelli et al., 2001). Mycoplasmas hosted by T. vaginalis have the privilege to evade host immune response, enhance T. vaginalis virulence and affect susceptibility to antibiotic treatment, thus complicating the infection eradication process (Dessi et al., 2005). In particular, a higher rate of phagocytic activity and amoeboid transformation, indicative of increased virulence, is observed in T. vaginalis isolates harboring M. hominis as compared with the mycoplasma-free isolates (Vancini et al., 2008). The endosymbiotic relationship may also alter the host inflammatory respond during trichomoniasis subsequently affecting disease severity (Fiori et al., 2013).

The broad term “mycoplasmas” collectively refers to both the Mycoplasma spp. and the Ureaplasma spp. The latter differentiate to Ureaplasma urealyticum and Ureaplasma parvumwhich mainly colonize the urogenital tract mucosal surfaces of sexually active women (McIver et al., 2009). U. urealyticum used to be categorized into biovars 1 and 2; however, according to phylogenetic analysis this taxonomic overlap has changed with the identification of the U. parvum formerly known as U. urealyticum biovar 1 (Kong et al., 1999). Both species have been controversial for their participation in human diseases and are generally considered as “low-grade pathogens” (Cox et al., 2016). The arrival of U. parvum at the forefront of research studies has raised questions regarding the virulence and pathogenicity of the microorganism formerly known as a non-pathogenic commensal of the male urethra. In fact, recent studies have proved the participation of the bacterium in pregnancy complications, intrauterine infections, vaginosis, and pelvic disease (McIver et al., 2009; Larsen and Hwang, 2010; Cox et al., 2016). Of great clinical significance is the association of preterm neonates ureaplasmal infection with adverse health outcomes predominantly including bronchopulmonary dysplasia, central nervous system deformities, and necrotizing enterocolitis (Pandelidis et al., 2013; Viscardi, 2014; Glaser and Speer, 2015). To date, Mollicutes including both Mycoplasma and Ureaplasma spp. are known to actively participate in sexually-transmitted infections though the exact role of the latter in trichomonas-associated infections is not widely investigated.

Treatment of Ureaplasma infection

A survey performed in several states between 2000 and 2004 detected tetracycline resistance in 45% of Ureaplasma isolates, indicating that the susceptibility of these organisms can no longer be assumed. [1] The degree of resistance may vary according to geographic area, patient population, and previous exposure to antimicrobial agents.

If tetracyclines are relied upon as first-line drugs, consider alternative agents in the event of treatment failures. In vitro susceptibility testing is sometimes indicated for Mycoplasma and Ureaplasma species recovered from a normally sterile body site, from hosts who are immunocompromised, or from persons who have not responded to initial treatment. Minimal inhibitory concentrations (MICs) for doxycycline are typically lower than those of tetracycline against these organisms.

Clindamycin is an alternative treatment for tetracycline-resistant M hominisbut is much less effective against Ureaplasma species. Macrolides, fluoroquinolones, or tetracyclines are the DOCs for Ureaplasma infections. Although tetracycline resistance is described in Ureaplasma species, high-level erythromycin resistance is uncommon, although it has been described in the United States and elsewhere due to ribosomal modification. A single 1-g dose of azithromycin is approved for treatment of urethritis due to Chlamydia trachomatis and works as well clinically as 7 days of doxycycline in persons with urethritis due to Ureaplasma species.

Clarithromycin, although active against Ureaplasma species in vitro at concentrations comparable to or lower than erythromycin, has not been approved for use in the treatment of urogenital infections.  Despite apparent in vitro susceptibility of Ureaplasma species to tetracycline or erythromycin, treatment of vaginal organisms with these agents is not always successful.

Fluoroquinolones are useful alternatives for treatment of certain infections caused by M hominis or Ureaplasma species within the urogenital tract and in some extragenital locations. Activity of quinolones is not affected by tetracycline resistance, making these drugs attractive alternatives for tetracycline-resistant M hominis or Ureaplasma infections. Levofloxacin and moxifloxacin have the greatest in vitro potency. In general, M hominis is more susceptible to quinolones in vitro than Ureaplasma species based on MICs. Fluoroquinolone resistance among M hominis and Ureaplasmaspecies has been reported in several countries, often in patients with prior exposure to these drugs, but the extent to which this occurs in the general population is unknown.

Most clinical trials for treatment of genitourinary infections focus primarily on other pathogens, such as C trachomatis and Neisseria gonorrhoeae. Few studies include microbiologic data specific to genital Mycoplasmaspecies, and no systematic comparative evaluations have been performed on treatment regimens for extragenital infections in adults or infections in neonates.

Treatment recommendations, including dosage and duration of therapy, are based largely on in vitro susceptibility data, outcomes of treatment trials evaluating clinical response to syndromes such as pelvic inflammatory disease and urethritis that may be due to genital Mycoplasma, and individual case reports. For infections such as urethritis that may be transmitted venereally, sexual contacts of the index case should also receive treatment.

Experience with Mycoplasma or Ureaplasma infections in patients who are immunocompromised, especially those with hypogammaglobulinemia (who have been studied most extensively), demonstrates that although Mycoplasma species are primarily noninvasive mucosal pathogens in healthy hosts, they have the capacity to produce destructive and progressive disease. Infections may be caused by resistant organisms refractory to antimicrobial therapy and may require prolonged administration of a combination of intravenous antimicrobials for several weeks or even months, intravenous immunoglobulin, and antisera prepared specifically against the infecting species. Even with aggressive therapy, relapses are likely. Repeat cultures of affected sites may be necessary to gauge in vivo response to treatment.

Isolation of M hominis or Ureaplasma species from neonatal pericardial fluid; pleural fluid; tracheal aspirate in association with respiratory disease; abscess material; CSF from those with pleocytosis, progressive hydrocephalus, or other neurologic abnormality; or blood justifies specific treatment in neonates who are critically ill when no other verifiable microbiologic etiologies of the clinical condition are apparent. Whether treatment should be given for a positive CSF culture when inflammation or other evidence of clinical illness is not observed should be handled on a case-by-case basis. Monitoring the patient, repeating the lumbar puncture, and reexamining for inflammation and organisms may be appropriate before initiating treatment because some cases may resolve spontaneously without intervention.

Parenteral tetracyclines are used most often to treat neonatal meningitis caused by either M hominis or Ureaplasma species, despite contraindications. Erythromycin for Ureaplasma species, clindamycin for M hominis are alternatives. Treatment of ureaplasmal respiratory infections in neonates with erythromycin or azithromycin may be effective in eradicating the organisms from the lower airways, but treatment failures are known to occur. No single drug is successful in every instance for eradication of these organisms from the CSF of neonates. Little clinical experience is available with new-generation macrolides such as azithromycin in the treatment of neonatal Ureaplasma infections, and no guidelines for their dosages or use in neonates are available.

Drugs such as the aminoglycosides and chloramphenicol sometimes demonstrate activity against genital mycoplasmas in vitro. However, their toxicities and the availability of better agents means they are not normally considered as suitable antimycoplasmal agents, with the exception of occasional use of chloramphenicol for treatment of systemic infections in neonates caused by M hominis or Ureaplasma species in the setting of tetracycline resistance or clinical failure with other agents.

PERSONAL PART

CHAPTER 5

MATERIAL AND METHODS

The study was performed during a sxi months period , janury – june 2018, by exmination of genital secretions and urine samples of women and men with clinical simptomatology of genital nfections

The study was realised in the microbiology departament of the Fculty of Medicine Constanta and Steaua Sudului Med laboratory Constanta

Colection of vaginal secretion

Materials required: • Gynecological valves (pair) or speculum • Microscope blades • Individually packed sterile buffer • Sterile compresses • gloves • Container for medical waste • Container with disinfectant solution for medical instruments • Cervical brush • Medium vial for Ag Mycoplasma / Ureaplasma purchased from the laboratory is stored in the refrigerator if not used on the day.

Collection is carried out on the gynecological table by staff trained by a specialist. Check whether the patient meets the preparation conditions for the genital secretion exam (48 hours without sexual intercourse, 7-10 days after the last menstrual period, not to be treated with antibiotics). [34]

The patient is invited to sit on the gynecological table as comfortable and relaxed as possible. If metallic valves are used, the patient is advised that the valves produce a slight feeling of discomfort. Introducing the metal valves or the speculum, the vaginal walls move away to express the cervix between the two valves. Remove the excess of secretion if necessary.

Using a sterile swab, which is then dumped into the medical waste container. Then with another sterile swab or a cervical brush it will be harvested as necessary: ​​the swab will rotate with fine movements, but with the care that the harvest is optimal; with the cervical toothbrush will also be grinded with fine movements and taking care not to cause injuries to the cervix or the walls of the vaginal mucosa. The buffer or brush should be placed in the MediCola vial for Ag Mycoplasma / Ureaplasma, taking care that the secretion is immersed in the transport medium and sent to the laboratory as soon as possible for examination. The barcode label sticks to the containers identical to the one on the application or other containers collected at the same time. [50]

Collection of uretral secretion

Materials required:

• Sterile swab and/ or sterile buffer packed individually

• Microscope slides: 2 for each sample

• gloves

•Sterile compresses

• Medium vials for Ag Mycoplasma / Ureaplasma, purchased from the laboratory is stored in the refrigerator if not used on the day.

Collection mode: It is recommended that the collection to be done by the urologist or gynecologist at the consulting room. If this is not possible harvestingcollection is done by the nurses trained in advance by the specialist doctor. It is insistently recommended that the patient should not have urinated at least 2 hours before harvesting. Collection is done with gloves. In men, the spontaneous urethral leakage or caused by mild pressure on the urethra is collected on the pad. If spontaneous leakage does not occur, the penis is caught between the medial finger and the annular ring and the index finger opens the urine. Besides sexual excitement and recent urination, urinary tract should not contain visible fluid. If outside of these two conditions the meat shows traces of fluid, it is taken off the swab. If fluids are seen in the urine, after brushing with the swab, the meat is imprinted on two microscope slides that have been removed beforehand with alcohol and dried. On these blades, the patient's name is usually written with the usual black pencil and sticks a bar code tag corresponding to the request and the product pad.

The lamellas are left to dry in a place protected from air drafts. They are sent to the laboratory together with the buffer inserted into the transport medium, especially for mycoplasms, taking care that the product is immersed in the transport environment. If no meat can be sampled, the intraurethral collection is performed with the buffer. The patient is lying down and cautions that harvesting involves an unpleasant maneuver. The swab is inserted into the gentle urethra with fine rotation movements up to a depth of about 2 cm. Upon retirement, all rotating movements are imprinted on the swab. This first buffer is discharged onto two microscope slides on which the name of the patient is written with the pencil and stick labels with the bar code corresponding to the request for the microscopic examination of mycoplasma. Repeat the harvest with a second buffer, this time intruding approximately 1 cm deeper than with the first buffer. [50]

In men, Mycoplasma and Ureaplasma can also be detected from sperm fluid. The sample is harvested after rigorous hygiene of the limb. The amount of sperm liquid is harvested directly into the sterile container (urocultor) – it is forbidden to use lubricants. The sterile urorecolators are usually used with the name, date, and patient registration number. The sample collected by the patient at home should be brought to the laboratory as soon as possible for examination.

Microscopic examination

For Trichomonas vaginalis I used the wet mount method, the specimen being mixed with saline and covered with a cover slip, then examined with a 40x magnification objective lens

Mycoplasma DUO

Mycoplasma DUO is a complete kit for the diagnosis of urogenital mycoplasms. Which makes possible the culture, identification, quantitative appreciation and testing of antibiotic susceptibility to Mycoplasma hominis and Ureaplasma spp.

Ureaplasma urealyticum was divided into two new species: Ureaplasma parvum and Ureaplasma urealyticum. They are considered together using Ureaplasmas spp.

PRINCIPLE:

The Mycoplasma kit comprises a selective culture broth with a strip of 22 assays.

The broth provides optimum growth conditions for mycoplasms (pH, sunblock, growth factor association).

If the culture develops, the specific sunstroke and the red phenol indicator in the broth (urea for Ureaplasma spp. And arginine for M. Hominis) will change their color due to the increase in pH.

The combination of three antibiotics and an antifungal ensures selectivity so that the contamination flora (Gram positive bacteria, Gram negative bacteria) present in the harvested product does not affect the test. After inoculation, the broth is pipetted onto the strip. The gallery simultaneously provides information about:

Identification; Quantitative Estimation and sensitivity to 9 antibiotics (doxycycline, josamycin, ofloxacin, erythromycin, tetracycline, ciprofloxacin, azithromycin, clarithromycin, pristinamycin). The quantitative estimation section determines whether the mycoplasms in the harvested product are greater than or equal to 104 UFC (colony forming units).

The kit can be used for the following products: • Urethral secretion, • Cervicovaginal Secretion, • Sperm (diluted 1/10 in physiological saline), • Other products (eg urine). Because mycoplasms have affinity for membranes in mucosal cells, it is important to squeeze the mucosa to harvest as many as possible cells. Harvesting will take place before any antibiotic is administered. After harvest the tanpone is immediately immersed in the R1 solution (stable nutrient broth, which inhibits bacterial growth).

Laboratory transport is carried out in R1 as quickly as possible under light shelter. Up to processing can be stored for 5 hours at room temperature or 48 hours in the refrigerator (4-8 ° C). After shaking, 3 ml of R1 will be transferred to the R2 container. R2 will be vortexed to completely dissolve the lyophilized content. (Fig. 1)

Fig.6.1: Collection and processing of the sample

Pipette 55 μl of broth into each well of the strip using an electronic pipette. Add 2 drops of mineral oil to each well. Cover the strip with the lid. Incubate 24 and 24 hours at 36 ° C. (Fig.)

Fig.2: Inoculation of the gallery

The color R2 is observed at 24 and 48 hours:

Yellow = negative,

Orange-red = clear Ureaplasma spp (Uu).

Slightly cloudy M. Hominis

The strip will be read at 24 hours – well no. 4 (Ureaplasmaspp> 104)

And 48 hours – the other wells

Fig.3. Stage Reading and species identification M. Hominis

Fig. 5 Identification of U.urealyticum

Fig. 6. Identification of both M.hominis and U.urealyticum

Figure 7.Susceptibility tests for identified species

Fig. 7. Stage V Interpretation of the sensitivity tests

Figure 9. Gallery

CHAPTER 6

RESULTS AND DISCUSSIONS

The patients belonged to different age groups ( Table 6.1.)

Table 6.1.Age characteristics of the patients

From a total of 108 samples of vaginal and cervical secretions from women presented in clinical ambulatory clinic with vaginitis, the following microorganisms were isolated in the 6-month period:

29 strains of Candida sp. (26,85%)

20 strains of Gram-negativ bacilli ( 18.51%),

12 strains of Gram-pozitive cocci ( 11,11%), and among them 5 cases Streptococcus agalactiae (beta-hemolytic streptococci group B) – 4.62 % and 7 straisn of Staphylococcus aureus ( 6.48%) .

12 of non-specific bacterial vaginosis ( 11,11%) .

Ureaplasma urealyticum was found in 9 samples ( 8.33%) , Mycoplasma hominis in 5 samples ( 4.62%), and Chlamydia trachomatis in 7 samples (6.48%).

Trichomonas vaginalis în 6 samples ( 5.55%).

Figure 6.2.Pathogenic species isolated from vaginal secretions

In the rest of the samples no micro-organisms suggestive of infection were isolated.

Non-specific bacterial vaginosis results from the relative lack of Lactobacillus saprophytic bacteria in the vagina, decrease of vaginal acidity and multiplication of anaerobic bacteria such as Mobiluncus, Gardnerella, Prevotella, and other anaerobic bacteria. Due to the very high incidence of Gardnerella vaginalis bacteria in the etiology of bacterial vaginosis, this is synonymous with some authors with Gardnerella vaginalis infection (15). This phenomenon is not well explained although it is associated with sexual activity. Approximately 50% of the bacterial vaginosis is asymptomatic.

In my study, among the patients with Trichomonas vaginalis, in 3 cases there were only Trichomonas infections, in two cases Trichomonas was associated with Candida albicans, and only in one case Trichomonas vaginalis was associated with Mycoplasma .

In other words, out of 5 cases on Mycoplasma hominis infection , in one case there was demonstrated mycoplasma and Trichomonas co-infection ( Figure)

The overall frequency of Mycoplasma and Trichmonas infetion was 1 in 108 patients with clinical symptomatology of vaginitis ( 0.92%) .

„The symbiotic relationship between T. vaginalis and Mycoplasma/Ureaplasma spp “ was already described by various authors [Anastasios Ioannidis, Panagiota Papaioannou], but more sensitive methods of detection can increase significantly the rate of detection.

Figure 6.3.Trichomonas and associations with other pathogens

In the group of patients, out of 108 women, there were 23 pregnant ones. In this group, I found Candida infections ( 16 cases) , Streptococcus agalactiae ( 3 cases) and 2 cases of Trichomonas vaginalis. Two women had no infection.

Figure 6.4. Results for pregnant women vaginal secretions

In the present study, the incidence of Trichomonas vaginalis infection in pregnant women was low, but Trichomonas infection is dangerous during pregnancy, because it increases the risk of premature rupture of membranes and premature birth. The infection can be transmitted to the fetus and is manifested as mild vaginitis.

The results should be evaluated considering that the wet mount , which is worldwide recognised as a traditional screening method of diagnosis of Trichomonas vaginalis infection, has a good specificity but a lower sensitivity, some authors indicating 58% [Wiese M]

. International organizations recommend for Trichomonas infected pregnant women and who show symptoms a single dose of metronidazole or tinidazole orally. The exception is pregnant during the first 12 weeks of pregnancy, to which these antibiotics are contraindicated.

Both pregnant women were in the third trimester, so with the possibility of treatment.

In pregnant women with asymptomatic infection, treatment with metronidazole is generally not recommended.

I found no case of association between Mycoplasma/Ureaplasma and Trichomonas vaginalis

Comparing our results with other studies, realised by wet mount and culture methods, that indicate the association betweem Trichomonas vaginalis in about 2% of the cases, we obtained a reduced rate of this co-infection. The colonisation of the genital epithelium is done independentely in the majority of the cases.

Also, the presence of other Mycoplasma species on the genital mucosa cannot be excluded [Martin DH].

Also, T.vaginalis is associated with Mycoplasma other anaerobic bacterial species (Parvimonas) and the decrease in lactobacilli number [Brotman]

T. vaginalis disrupts the urogenital epithelia and produce vaginal and endocervical inflammation in women and urethral inflammation in men with trichomoniasis likely contribute to enhance the transmission of viral infections. [7]. Also, T. vaginalis infection produce punctate mucosal hemorrhages of the genital mucosa compromising the mechanical barrier to other pathogens

In this case bacterial vaginosis and Trichomonas vaginalis benefit both from Metronidazol therapy.[Ratod SD].

For Mycoplasma and Ureaplama sensitiviy tests can be performed. The Mycoplasma DUO kit , by the microdilution method , are testes to macrolides, cyclines ( doxicycline and fluoroquinolones) .These antibiotics are indicated ain all the guides as firlst line therapy in genital infections

The results obtained by testing sensitivity to antibiotics of isolated Mycoplasma and Ureaplasma strains are presented in the table below ( table 6.2.):

Table. 6.2.Sensitivity tests results

Doxycycline (Vibramycin) belongs to the Tetracycline class: broad-spectrum bacteriostatic. Inhibits protein synthesis by binding to the 30S subunit. It works on: gram positive and Gram negative bacteria, Vibrio, Shigella, Treponema pallidum, Mycoplasma, Ricketsia, Chlamydia.

Roxithromycin, Azithromycin, Josamycin are part of the macrolide class: antibiotics with a bacteriostatic effect, which act by binding to the 50S subunit of ribosomes. Spectrum of action is broad, Penicillin type, to which are added Haemophyllus, Bordetella, Chlamydia, Mycoplasma and Ureaplasma, anaerobes: Peptostreptococcus, Actinomyces, Clostridium, Bacteroides, Atypical mycobacteria, Ricketsii, Lysteria. Resistance to macrolides occurs as a result of the bacterial wall permeability of the antibiotic. Side effects are gastrointestinal: nausea, vomiting, abdominal pain. Pristinamycin is a macrolide derived class with bactericidal action. The mechanism of action is identical to that of macrolides

The results of the tests showed that the best sensitivity was for Josamycin and Pristina mycin, followed by Azithromycin and Doxiclin. All these drugs can be indicated in the discussed cases.

The results for male patients

Among the 19 men with symptomatology of urethritis, I found Staphylococcus epidermidis in 4 cases, Ureaplasma urealyticum in 2 cases, Neisseria Gonorrhoeae in 2 cases and Candida albicans in 5 cases and Trichomonas vaginalis in one case. Trichomonas vaginalis was found in the urine specimen and not in the urethral secretion

In two cases Candida and Staphylococcus were isolated in the same patient

Figure 6.5. Distribution of cases in men with urethritis

Although the number of men presentation in the laboratory was segnificantly smaller than the women, we can notice that in man the association Staphylococcus epidermidis and Candida is really much frequent than Trichomonas or Mycoplasma and Ureaplasma.

CONCLUSIONS

From a total of 108 samples of vaginal and cervical secretions from women presented in clinical ambulatory clinic with vaginitis, Trichomonas vaginalis was found în 6 samples ( 5.55%), Ureaplasma urealyticum was found in 9 samples ( 8.33%) , and Mycoplasma hominis in 5 samples ( 4.62%).

In my study, among the patients with Trichomonas vaginalis, in 3 cases there were only Trichomonas infections, in two cases Trichomonas was associated with Candida albicans, and only in one case Trichomonas vaginalis was associated with Mycoplasma .

There were 23 pregnant women, and in this group, I found 2 cases of Trichomonas vaginalis.

The results should be evaluated considering that the wet mount , which is worldwide recognised as a traditional screening method of diagnosis of Trichomonas vaginalis infection, has a good specificity but a lower sensitivity, some authors indicating 58%

The overall frequency of Mycoplasma and Trichmonas infetion in female patients with clinical symptomatology of vaginitis was 0.92% .

The symbiotic relationship between T. vaginalis and Mycoplasma/Ureaplasma spp is described by various authors but more sensitive methods of detection can increase significantly the rate of detection.

The number of men presentation in the laboratory was segnificantly smaller than the women, stll we can notice that in man the association Staphylococcus epidermidis and Candida is really much frequent than Trichomonas or Mycoplasma and Ureaplasma.

The results of the tests showed that the best sensitivity fwas for Josamycin and Pristina mycin, followed by Azithromycin and Doxiclin ( for Mycoplasma and Ureaplasma). They can be associated with Metronidazole for Trichomonas co-infection

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