Chlamydial infections

Dr.Ramesh.R

History
The

word Chlamydia is derived from Greek, which means over coat i.e inclusion bodies coating the host cell nucleus Trachoma was described in Egyptian papyri LGV was described in the 8th century The organism was first isolated from patients with LGV in 1930s Major breakthrough in 1960s was the development of tissue culture isolation procedures

Classification

Kingdom: Phylum: Order: Family: Genus:

Bacteria Chlamydiae Chlamydiales Chlamydiaceae Chlamydia

Introduction
They

are nonmotile, obligatory intracellular bacteria with unique biphasic developmental cycle Tropism for squamous epithelial cells and macrophages Filterability and failure to grow in cell free media Absence of peptidoglycan in the cell 4 wall

Cont.
Chlamydiaceae

show less than 10% 16SrRNA gene diversity and <10% 23SrRNA gene diversity The genome size of the Chlamydiaceae ranges from 1.0 to 1.24 Mbp with a G+C content of about 40% LPS has low endotoxic activity compared to other bacteria due to unusual long chain fatty acids in lipid A

Cont Major outer membrane protein(MOMP) is coded by ompA gene

Cysteine

rich OMP2(OmcB) forms disulfide cross linkages with MOMP domains

Provides

osmotic stability to elementary bodies

OmcB

Binds

to heparin and related to mammalian host cell adhesion and entry

Cont
Chlamydia

lack enzymes of the electron transport chain and so require ATP and nutrient resources from host cell Takes on two forms in its life cycle: Elementary body (EB) Reticulate body (RB)

Structure and chemical composition

a

non-replicating, infectious particle called the elementary body (EB, measuring 200-300nm)

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an intracytoplasmic form called the reticulate body (RB, measuring 5001000nm )

Cont
The

genus Chlamydia contains four species 1.C trachomatis 2.C psittaci 3.C pneumoniae 4.C pecorum Species differentiation is based on growth characteristics, nucleic acid profile, antigens, plasmids and nature of the inclusion body

Characteristics of Chlamydiae Property C . trachomat C . psitta C . pneumoniae

Elementary Round is body Inclusion Round, vacular body Glycogen Yes inclusions Plasmid DNA Yes Susceptibilit Yes y to Release of EB 72-96 hours sulfonamides Round ci Variable, dense No Yes No 48hours Pear -shaped Round, dense No No No yes

Cont
Species
C.psittaci Many C. Trachoma trachomatis D-K LGV C.pneumoniae TWAR

Biovars

Method of Natural Aerosol, Birds, sheep, spread host

sexual(in Fomites, animal flies. hosts) , hand Sexual to eye, neonatal Sexual Aerosol cat etc Humans Humans Humans

Humans

Antigens
Genus

-specific antigens: heat-stable LPS as an immunodominant component. Antibody to these antigens can be detected by CF and immunofluorescence Species-specific or serovarspecific antigens Antigens are mainly outer membrane proteins(MOMP). Specific antigens can best be detected by immunofluorescence,particularl y using monoclonal antibodies.

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Pathogenesis
An

elementary body attaches to a susceptible epithelial cells by using heparin sulfate as a bridge Enters the epithelial cell by receptor mediated endocytosis The EB undergoes reorganization into larger replicative form, the reticulate body The RBs are osmotically unstable and incapable of infecting another cell

Cont RBs synthesize their own DNA, RNA and protein but lack the necessary metabolic pathways to produce high-energy phosphate compounds. They divide by binary fission The organisms are released from the infected cells by cell lyses(C.psittaci) or exocytosis(C.trachomatis)

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Developmental cycle

16

species that cause disease in humans

Species Disease Trachoma, NGU, PID, conjunctivitis, Infant pneumonia, Pharyngitis, LGV bronchitis, pneumonia Psittacosis

C. trachomatis 2 biovars TRIC C. pneumoniae LGV C. psittaci

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Chlamydia trachomatis
The

name trachoma comes from 'trakhus' meaning rough which characterizes the appearance of the conjunctiva. It is one of the commonest cause of preventable blindness and impose a significant burden on humans globally It has been estimated that worldwide 500 million people are affected by ocular trachoma with some 7-9 million blind as a result

Cont.
Contains

18 serovars Serovars A,B,Ba and C are associated with trachoma Serovars D through K including the serovars Da, Ia and Ja are associated with genital tract diseases(most common STD) Serovars L1, L2,L2a, L2b and L3 are associated with LGV(STD)

Risk Factors
Adolescence New

or multiple sex partners History of STD infection Presence of another STD Oral contraceptive user Lack of barrier contraception

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Clinical Syndromes Caused by C . trachomatis

Local Infection Complication Reiters syndrome Epididymitis Endometritis Salpingitis Perihepatitis Reiters syndrome Chronic lung disease Sequelae Chronic arthritis (rare) Infertility (rare) Infertility Ectopic pregnancy Chronic pelvic pain Chronic arthritis Rare, if any (rare)

Men

Conjunctivitis Urethritis Prostatitis Conjunctivitis Urethritis Cervicitis Proctitis Conjunctivitis Pneumonitis Pharyngitis Rhinitis

Women

Infants

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Clinical syndromes and presentations

trachomatis Trachoma
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C.

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Cont

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C . trachomatis Complications in Women

Pelvic

Inflammatory Disease (PID) Salpingitis Endometritis Perihepatitis (Fitz-Hugh-Curtis Syndrome) Reiters Syndrome
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C . trachomatis Infections in Infants

Perinatal

clinical manifestations: conjunctivitis

Inclusion Pneumonia

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Chlamydia psittaci
The

term psittacosis is Greek word, meaning parrot It is common in birds and domestic animals The infection is generally spread by the respiratory route and direct contact The severity of the disease ranges from inapparent or mild disease to a fatal systemic illness with prominent respiratory symptoms

Chlamydia pneumoniae ( twar )

Among

Chlamydia species It is the most common cause of human infections The TWAR is the only serovar of C.pneumoniae It is transmitted from person to person by respiratory tract secretions The clinical manifestations of an acute C.pneumoniae infection can range from asymptomatic to life threatening illness

Cont
A

special association of C.pneumoniae infections with asthma has been proposed studies have associated coronary artery disease and atherosclerosis with antibody to C.pneumoniae organism has been demonstrated in atheromatous plaques of coronary arteries

Seroepidemiologic

The

Laboratory diagnosis
Microscopic

demonstration of inclusion or elementary bodies Isolation of Chlamydia Demonstration of Chlamydial antigens Nucleic Acid Amplification Tests (NAATs) serology

 As

they are obligate intracellular pathogens, specimen collection should include the host cells that harbor the organisms Specimens should be forwarded to the laboratory with in 24 h in a special Chlamydial transport medium such as 2 sucrose phosphate or sucrose phosphate glutamate supplemented with fetal calf serum(5-10%), gentamicin(10ug/ml), vancomycin(25100ug/ml) and amphotericin B(2ug/ml) or nystatin(25u/ml)

Samples collection , transport and storage

Cont.. During transportation temp should be maintained at 4-80C

Specimens

can be stored at -700C when there is a delay in processing more than 24 h for culture should not be stored at -200C or in frost free freezers

Specimens

Cont
Cervical

and ocular specimens are collected by scrapings with the help of Dacron tip or rayon swabs with an aluminum or plastic shaft tips made of calcium alginate with wooden shafts may inhibit the chlamydiae

Swab

Chlamydia trachomatis
First

void urine ( first 10-30ml) and vulvovaginal swab specimen are useful for NAATs may be stored at ambient temperature and should be processed with in 24h to avoid denaturation of chlamydial DNA

Samples

Cont. Traditional sites for specimen collection in genital tract infection involve the endocervixin females and the urethra in males Purulent discharges/mucus have to be cleaned with sterile saline before swab is inserted 1 to 2 cm in to the cervical os past the squamo-columnar junction and rotated more than two times and removed without touching the

Collection of conjunctival scrapings

Anaesthetize

the conjunctiva with anesthetic eye drops Clean the mucus/discharge with sterile saline Use thin blunt end of spatula to scrape whole conjunctiva Spread the specimen evenly on a slide Air dry the smear and fix it with methanol for Giemsa staining or acetone for flurochrome staining

Cont.
Urethral

specimens from males are collected by placing a dry swab 34cm into the urethra and rotating more than twice Urination prior to specimen collection may reduce test sensitivity by washing out infected columnar cells In salpingitis, LGV, samples may be collected by needle aspiration of the involved fallopian tube and buboes

Cont
Specimens

for C. pneumoniae include sputum, bronchoalveoalar lavage fluid, nasopharyngeal aspirates, throat washings and throat swabs Specimens for C. psittaci include sputum, BAL fluid, pleural fluid, blood and tissue biopsy Liquid specimens are collected in a transport medium at a specimen to medium ratio of 1:2

Cont. Tissues taken at autopsy or sputum should be homogenized or shaken with glass beads in sucrose potassium glutamate Specimens collected by swabs should be shaken for 1min on a vortex mixer It is centrifuged at 500g for 5 min to remove cellular debris and stored at -600C until tested

Presumptive diagnosis
A

presumptive diagnosis of acute chlamydial infection in male patients is made when 1. 5 pus cells/hpf in Gram stained urethral smear and no intracellular Gram negative diplococci or 2. More than 20 pus cells in a first voided urine specimen

Identification of chlamydiae trachomatis in giemsa and iodine stain

Stain Reticulate Elementary Host bodies bodiesred, nucleus Giemsa Blue MauvePink-mauve stain mauve, less dense closely inclusion bodies containing glycogen packed stains brown with iodine stain

Staining properties
EBs

stain purple with Giemsa stainin contrast to the blue of host cell cytoplasm. RBs stain blue with Giemsa stain. The Gram reaction of chlamydiae is negative or variable and is not useful in identification. Staining with iodine can distinguish between inclusion bodies of C trachomatis and C psittaci, as only the former contain glycogen Inclusions stain brightly by immunofluorescence ,with group44 specific,species-specific, or serovarspecific antibodies.

Nucleic acid amplification tests

Tests

of choice for diagnosis of genital C.trachomatis infections in routine clinical laboratories NAATs may be used for screening programs PCR and strand displacement amplification(SDA) assay amplify nucleotide sequences of the cryptic plasmid Transcription mediated amplification based assays target specific sequences of the 23SrRNA

Direct fluorescence assay ( DFA )

Fluorescein

isothiocyanate conjugated monoclonal antibodies directed against C trachomatis specific epitope of the MOMP is used They are based on detecting EBs in smears The procedure offers rapid diagnosis(30 min) It has sensitivity of 75-85% and specificity of 98-99%

Enzyme immunoassay
Monoclonal

or polyclonal antibodies are used to detect chlamydial LPS It has sensitivity of 62-72% when culture is used as reference standard There is a risk of false positive results due to cross reaction with LPS of other microorganisms

Isolation
Historically the gold standard Sensitivity and specificity are

close to 100% Use in legal investigations Not suitable for widespread screening Disadvantages include technical complexity, long turnaround time, and stringent requirements related to collection, transport and storage of specimens

Isolation procedures
C.pneumoniae

and C.trachomatis are BSL-2 organisms, where as C.psittaci is a BSL-3 organism Transmission of the organisms from patient specimens or infected cell cultures may occur through aerosols, splashes in to mucous membranes and hand to face actions

Cont. Preventing laboratory acquired infections include the use of gloves, alcohol based hand disinfectants, safety centrifuge caps and face protection Laboratory infection with C.trachomatis usually manifest as follicular conjuctivitis The LGV strains are more invasive and causes severe pneumonia and lymphadenitis

Cont. McCoy and HeLa 229 cells are most commonly used for C.trachomatis HL and Hep-2 cells are used for the isolation of fastidious C.pneumoniae Host cells are plated either onto 12mm glass cover slips contained in 15mm diameter disposable glass vials or in tissue culture plates (6,12, 24 well)

Cont. The cells are seeded in a conc of 1x105 to 2x105 cells/ml Confluent monolayer formation occurs with in 24-48 h Specimens are thoroughly vortexed with glass beads in a tightly closed vials to facilitate the release of chlamydiae and inoculated onto the cell monolayers(with in 24 h of confluency)

Cont. The inoculated specimen is centrifuged on to the cell monolayer's at 900 to 3000 X g for 1 h at 22-350C Cells are incubated at 350C for 1-2 h to allow uptake of chlamydiae Then chlamydial isolation medium consisting of the cell culture medium supplemented with fetal calf serum(10%), L-glutamate(2mM), cycloheximide, gentamicin, vancomycin and amphoterecin B is added and incubated at 350C in 5%CO2 for 48-72h

 It

involves demonstration of intracytoplasmic inclusions by fluorescent antibody staining Cultures can be screened by using fluorescein isothiocyanate conjugated anti-LPS antibody which recognizes all chlamydiae Other methods of staining include Giemsa and iodine staining

Identification

Cont Confirmation of positive genital cultures can be done by the use of a C.trachomatis MOMP specific monoclonal antibody Identification of replicating chlamydiae can also be done by fluorescence in situ hybridization using fluorescent labeled oligonucleotide probes complementary to order, genus and species specific target sites on the chlamydial 16S rRNA

Isolation using chick embryo ( 6 - 8days )

Inoculate

0.2-0.5ml suspension of specimen treated with gentamicin(500ug/ml) and nystatin(100u/ml) in to the yolk sac of each six embryos Incubation at 350C Eggs candled daily and discard any embryo which die with in 48 hours

Cont. Harvest yolk sacs of the embryo after 13 days of inoculation Impression smears are prepared from the yolk sac and stained with Giemsa or Gimenez Observe for the elementary bodies The yolk sac method is used for preparing antigens for the microimmunofluorescence test

Serological tests
Complement Microimmunofluorescence Enzyme

fixation test test

immunoassay

Complement fixation test

It

is based on antibody reactivity to the Chlamydia LPS antigen It may be useful in diagnosing symptomatic LGV and psittacosis. A titer of >256 strongly supports the clinical diagnosis of LGV It is not recommended for the diagnosis of C.pneumoniae, trachoma, inclusion conjunctivitis due to cross reactivity and low sensitivity

Microimmunofluorescence test
It

is the method of choice for serodiagnosis of chlamydial infections Species and serovar specific antibodies can be detected It allows quantitative detection of IgM and IgG antibodies, helpful in distinguishing recent from past infections

Cont It is performed using purified formalinized EBs of representative serovars of C. trachomatis, C.psittaci and C.pneumoniae that are dotted in a specific pattern onto glass slides Serial dilutions of patient sera are placed over the fixed antigen dots and incubated The bound antibody is detected with fluorescein- conjugated anti-IgG or anti-IgM antibody Fluorescence is read by UV microscopy

Cont It is the test of choice for C.trachomatis pneumonitis in infants(IgM titer of >32) and LGV(IgG titer >128)
Paired

sera demonstrating at least a fourfold rise in titer and single serum samples with IgM >16 and/or IgG >512 is also useful in diagnosing acute C.pneumoniae

Immunochromatographic tests
They Antigens

are sensitive, specific and rapid tests are extracted from the endocervical, urethral swabs or urine and identified min results can be read after 10

Test

Antimicrobial susceptibilities and treatment

It

has little clinical utility as in vitro resistance does not correlate with the patients clinical outcome It requires growing the organisms in epithelial cells cultured in medium containing increasing conc of antibiotics Cells are stained with an FITC-labeled antichlamydial antibody and the lowest concof antibiotic that prevents the inclusion formation after 48 h of incubation is reported

Treatment of Uncomplicated Genital Chlamydial Infections

recommended regimens Azithromycin 1 g orally in a single dose, OR

Doxycycline

CDC -

7 days

100 mg orally twice daily for

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Alternative regimens Erythromycin base 500 mg orally 4 times a day for 7 days, OR
Erythromycin

ethylsuccinate 800 mg orally 4 times a day for 7 days, OR 300 mg orally twice a day for 7 days 500 mg orally once a day for 7 days

Ofloxacin

Levofloxacin

Repeat Testing after Treatment

Pregnant

women Repeat testing, preferably by NAAT, 3 weeks after completion of recommended therapy Non-pregnant women Test of cure not recommended unless compliance is in question, symptoms persist, or re-infection is suspected Repeat testing recommended 3-4 months after treatment, especially adolescents due to high prevalence of repeated infection

Prevention

66

Screen for Chlamydia

Most Screening

infections are asymptomatic.

can reduce the incidence of PID by more than 50%. decreases the prevalence of infection in the population and reduces the transmission of disease.
67

Screening

Screening Recommendations : Non - pregnant Women

Sexually

active women age 25 years and under should be screened annually. Women >25 years old should be screened if risk factors are present. Presentation for induced abortion

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 Sex

partners should be evaluated, tested, and treated if they had sexual contact with the patient during the 60 days preceding the onset of symptoms or diagnosis of Chlamydia. recent sex partner should be evaluated and treated even if the time of the last sexual contact was >60 days before symptom onset or diagnosis. of therapy to sex partners by heterosexual male or female patients 69 might be an option.

Partner Management

Most

Delivery

Chlamydia psittaci
Treatment

of infected birds with tetracycline, chlortetracycline, or doxycycline for at least 45 consecutive days Prophylactic treatment of all imported birds Quarantine of all imported birds for 30 days Environmental sanitation Avoiding close contact with birds and domestic animals

THANK YOU

REFERENCES Ananthanarayan and Panikers text book of microbiology Topley and Wilsons microbiology and microbial infections Manual of clinical microbiology, 9th edn. Mackie and McCartney, Practical medical microbiology

Transmission
Transmission

is sexual or vertical Highly transmissible Incubation period 7-21 days Significant asymptomatic reservoir exists in the population Re-infection is common Perinatal transmission results in neonatal conjunctivitis in 30%-50% of exposed babies 73

Transmission
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C. pneumoniae
Person-to-person

spread by inhalation of infectious aerosols. No animal reservoir

C.psittaci
Infectionacquiredbycontactwith infectedbirdoranimal(may appearhealthy). Persontopersoninfectionvery uncommon.

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Virulence factors
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C. pneumoniae
Intracellular

replication; prevention of phagolysosome fusion; ability to infect and destroy ciliated epithelial cells of respiratory tract,smooth muscle cells,endothelial cells,and macrophages; extracellular survival of infectious EBs.

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Clinical syndromes
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C. pneumoniae
Bronchitis Pneumonia Sinusitis Pharyngitis atherosclerosis

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Diagnosis
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Sera and tears from infected humans are used to detect anti-Chlamydia antibodies by the complement fixation or microimmunofluorescence tests. The latter is useful for identifying specific serotypes of C trachomatis. Fluorescent monoclonal antibodies are used to stain C trachomatis elementary bodies in urethral and cervical exudates.

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Diagnosis
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It is possible to diagnose C trachomatis in tissue biopsy specimens by in situ DNA hybridization with cloned C trachomatis DNA probes. DNA from C trachomatis isolates can be examined by restriction endonuclease analysis. The DNA cleavage pattern of C trachomatis isolates differs greatly from that of DNA from C psittaci isolates. DNAs of the agents of trachoma and lymphogranuloma venereum differ in their cleavage patterns, and this allows identification of the biovars

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Diagnosis
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Chlamydia pneumoniae DNA has 10 percent homology with C trachomatis or C psittaci; C pneumoniae isolates have 100 percent homology. Chlamydia pneumoniae isolates can be diagnosed by hybridization with a specific DNA probe that does not hybridize to other chlamydiae. Two additional serologic tests are in use: the microimmunofluorescence test with C pneumoniae-specific elementary body antigen, and the complement fixation test, which measures Chlamydia

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Taxonomy

Chlamydia trachomatis is an important pathogen of humans and 1 of 4 species within the genus C. trChlamydia trachomatis is an important pathogen of humans and 1 of 4 species within the genus C. trachomatis (natural host: humans) C. pneumoniae (natural host: humans) C. psittaci (natural host: birds, lower mammals) C. pecorum (natural host: sheep, cattle, and swine) 4 biovars of C. trachomatis Murine biovar Swine biovar Lymphogranuloma venereum (LGV) biovar Trachoma biovar achomatis (natural host: humans) C. pneumoniae (natural host: humans) C. psittaci (natural host: birds, lower mammals) C. pecorum (natural host: sheep, cattle, and swine) 4 biovars of C. trachomatis Murine biovar Swine biovar Lymphogranuloma venereum (LGV) biovar Trachoma biovar

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Growth and synthesize ATP metabolism Unable to

and depend on the host cell for energy requirements .

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Grow in cultures of a variety of eukaryotic cell lines

McCoy cells are used to isolate chlamydiae

C pneumoniae grows better in HL or Hep - 2 cells . All types of chlamydiae proliferate in embryonated eggs , particularly in the yolk sac .

The replication of chlamydiae can be inhibited by many antibacterial drugs .

Cell wall inhibitors ( penicillins ) result in the production of morphologically defective forms but are not effective in clinical diseases . 81 Inhibitors of protein synthesis ( tetracyclines , erythromycins ) are effective in

Classification C trachomatis
Biovar

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trachoma Biovar lymphogranuloma venereum Biovar mouse

C

pneumoniae C psittaci C pecorum

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Who is at risk?

C. trachomatis
People

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with multiple sexual partners. Homosexuals,who are more at risk for LGV. Newborns born of infected mothers. Reiters syndrome: young white men. Trachoma:children,particularly those in crowded living conditions where sanitation and hygiene are poor.

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Molecular Aspects
It

cannot synthesize ATP, so it needs living host cells to survive

Cannot grow on artificial media

Two

part life cycle

Non-replicating, infectious elementary bodies (analogous to a spore) Replicating, non-infectious reticulate body (replicate inside host cell, in a membrane-bound inclusion) When the bacterium gains entrance to the host cells elementary bodies germinate into reticulate bodies

http://www.healthofchildren.com/images/gech_0001_0004_0_img0276.jpg

Pathogenicity

Trachoma (conjunctivitis)
Most

common cause of blindness in the world Direct contact with infected eye discharge or flies that have trouched an infected person Causes the inflammation of the eye Eyelids turn inwards so the eye lashes rub against the cornea causing scar tissue to form Irreversible blindness

Characteristics
Rod-shaped or cocciod Obligate intracellular parasites Aerobic Gram negative but difficult to stain Cell Wall lipopolysaccharides form the outer membrane, not peptidoglycan. Forms elementary bodies Non-motile 37C, mesophile G+C Content - 41.3%

Pathogenesis
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EBs attach to the microvilli of susceptive cells. Penetration into the host cell via endocytosis or pinocytosis and forming phagosomes Fusion of lysomes with the EB-containing phagosome are inhibited EBs reorganize into the metabolically active RBs

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Transmission C. trachomatis
Sexually Infected

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transmitted;most frequent bacterial pathogen in united states. patients , who may be asymptomatic. through break in skin or membranes. to new born at birth.

Inoculation Passage

Trachoma

spread to eye by means of 88 contaminated hand,droplets,clothing, and flies.

C. trachomatis Syndromes Seen in Men or Women

Non-LGV serovars
Conjunctivitis Proctitis Reiters

Syndrome

LGV serovars
Lymphogranuloma

venereum

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C. trachomatis Infections in Children

Pre-adolescent males and females:

Urogenital

infections

Usually asymptomatic Vertical transmission Sexual abuse

90

Virulence factors

C. trachomatis
Intracellular replication, prevention of phagolysosomal fusion, survival of infectious EBs as a result of crosslinkage of membrane proteins.

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Pathogenicity
Transmitted through direct contact between infected membranes Genital tract infection
Asymptomatic in most cases Symptoms Painful urination Lower abdominal pain Unusual Discharge

If left untreated, common cause of infertility Newborns can contract the disease from infected mothers

Diagnosis
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Chlamydia

trachomatis can be identified microscopically in scrapings from the eyes or the urogenital tract. bodies in scraped tissue cells are identified by iodine staining of glycogen present in the cytoplasmic vacuoles in infected cells.

Inclusion

93

Cont
To isolate the agent, cell homogenates that contain the chlamydial elementary bodies are centrifuged onto the cultured cells (e.g., irradiated McCoy cells). After incubation, typical cytoplasmic inclusions are seen in the cells stained with Giemsa stain or iodine.

Treatment of Chlamydial Infection in Pregnant Women

CDC-recommended regimens
Azithromycin 1 g orally in a single dose, OR Amoxicillin 500 mg orally 3 times a day for 7 days

Alternative regimens
Erythromycin base 500 mg orally 4 times a day for 7 days, OR Erythromycin base 250 mg orally 4 times a day for 14 days, OR Erythromycin ethylsuccinate 800 mg orally 4 times a day for 7 days, OR Erythromycin ethylsuccinate 400 mg orally 4 times a day 95 for 14 days, OR

Treatment of Neonatal Conjunctivitis and/or Pneumonia

CDC-recommended regimen Erythromycin base or ethylsuccinate 50 mg/kg/day orally divided into 4 doses daily for 14 days

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Treatment of Chlamydial Infection in Children

Children who weigh <45 kg: Erythromycin base or ethylsuccinate 50 mg/kg/day orally divided into 4 doses daily for 14 days

Children who weigh 45 kg, but are <8 years of age: Azithromycin 1 g orally in a single dose

Children 8 years of age: Azithromycin 1 g orally in a single dose, OR Doxycycline 100 mg orally twice a day for 7 days

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Treatment of Lymphogranuloma Venereum (LGV)

CDC-recommended regimen Doxycycline 100 mg orally twice a day for 21 days

Alternative regimen Erythromycin base 500 mg orally 4 times a day for 21 days

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Diagnosis

Non-Amplication Tests
Direct
Detects

fluorescent antibody (DFA)

intact bacteria with a fluorescent antibody Variety of specimen sites

Enzyme

immunoassay (EIA)

Detects

bacterial antigens with an enzymelabeled antibody

Nucleic

acid hybridization (NA probe)

Detects

specific DNA or RNA sequences of C. trachomatis and N. gonorrhoeae

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Serology
Rarely used for uncomplicated infections Comparative data between types of serologic test are lacking Criteria used in LGV diagnosis

Complement

fixation titers >1:64 can support diagnosis in the appropriate clinical context Serologic test interpretation for LGV is not standardized

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Cont
Both the particles are suspended in 3-5% normal yolk sac or egg albumin glycerol The antigens are spotted with fine pen nibs on clean glass microscopic slides Normal yolk sac 5% is spotted as controls The slides are air dried and fixed in acetone 10ul of serum/secretions are placed over each group of antigens

Cont..
Fluorescein-isothiocyanate conjugated antispecies immunoglobulin is placed over each spot Slides are then air dried and covered with glycerol based mounting fluid and a cover slip Fluorescence is read by UV microscopy

Sample collection
Smear specimens should be obtained with a conjunctival spatula. NEVER USE A SWAB (soft-tipped applicator) TO OBTAIN A SMEAR FOR CYTOLOGY After applying topical . anesthetic, specimens are directly collected by firmly scraping the exposed conjunctiva. These specimens are transferred to glass microscope slides, air-dried, and allowed to remain at room temperature. Viral transport media are not suitable substitutes, since these usually contain penicillin.

Cont..

Collected samples are placed in 2.0 ml of chlamydial transport medium. We have had great success with Bartels ChlamTrans Chlamydial transport medium and recommend its use. DO NOT USE VIRAL TRANSPORT MEDIUM THAT MAY CONTAIN ANTIBIOTICS THAT INHIBITS CHLAMYDIA GROWTH IN CELL CULTURE. Chlamydia is very fastidious and will not survive unless refrigerated (short-term) or frozen (longterm) (-75 degrees C).

Cont..

Smear specimens should be obtained with a conjunctival spatula. NEVER USE A SWAB (soft-tipped applicator) TO OBTAIN A SMEAR FOR CYTOLOGY After applying topical . anesthetic, specimens are directly collected by firmly scraping the exposed conjunctiva. These specimens are transferred to glass microscope slides, air-dried, and allowed to remain at room temperature

Examples of specimens that are generally not acceptable for testing by nonculture methods are vaginal, rectal, nasopharyngeal, or female urethral specimens. In cases of suspected sexual assault or abuse, only culture tests should be used regardless of the specimen site A specimen would be determined to be inadequate if any one of the following conditions exist: no cellular components, no columnar or metaplastic cells, or the presence of only squamous epithelial cells or PMNs. Detergent-based transport media such as those conventionallyused for DNA probe or DNA amplification testswill lyse chlamydial elementary bodies and columnar cells and cannot be used for assessment of specimen adequacy

Currently, CDC recommends that culture be used for the detection of C. trachomatis in urethral specimens from women and asymptomatic men, nasopharyngeal specimens from infants, rectal specimens from all patients, and vaginal specimens from prepubertal girls. In cases of suspected sexual abuse or assault, only culture tests should be used to diagnose C. trachomatis infection

C.trachomatis in pap smear(H & E stain )

Cont. Repeat testing of all women 3-4 months after treatment for C. trachomatis infection, especially adolescents.
Repeat

testing of all women treated for C. trachomatis when they next present for care within 12 months.