CONTRIBUTION OF IMMUNOASSAY (WIDAL TEST / TYPHI DOT TEST) IN EMERGENCE OF ANTI MICROBIAL RESISTANCE (AMR)

WHAT IS TYPHOID FEVER?

Typhoid and Para typhoid fever is bacterial infection caused by SALMONELLA ENTERICA SEROVARS TYPHI and PARA TYPHI (S. Para typhi) respectively. Typhoid fever is also known as enteric fever or simply called as typhoid. It is estimated to cause millions of typhoid infections and death globally each year. It is an acute oral-fecal transmitted life threatening and systemic febrile illness. Children and immunocompromised patients are the most vulnerable population. It is common bacterial infection in low- or middle-income country which is characterized by high grade fever in step- ladder pattern that is rise and fall alternatively, chills, myalgias (pain in muscles), altered bowel habits (constipation or diarrhea), headache, lethargy, cough stomach ache and rose-colored spots development in few cases. Fatal complications of disease include intestinal perforation or GI hemorrhages, encephalitis, liver damage, inflammation of heart and cranial neuritis which exhibited in those patients whom anti-microbial treatment is not begin timely. Largest numbers of typhoid cases are being reported in South Asia (India, Pakistan and Bangladesh). Pakistan is facing high incidence rate of typhoid fever. Typhoid fever is an endemic in Pakistan and shows it sporadic or scattered occurrence (occur in few places and not occur at regular intervals).

CLASSIFICATION OF SALMONELLA BACTERIA

Salmonella is the genus which is divided into 2 species. Salmonella species are gram negative flagellated facultatively anerobic bacilli which is characterized by O (somatic antigen), H (flagellar antigen) and Vi antigen. It belongs to Enterobacteriaceae family. Salmonella species are as follows,
1. Salmonella Enterica
2. Salmonella Bongori
Among these 2 species Salmonella Enterica is further sub divided into 6 sub-species which includes 2500 serotypes. Salmonella serotypes can be divided as
• Typhoidal serotypes
It is human restricted serotype. It is not found in animal. Require prompt antibiotic treatment. It is systemic, complicated and life-threatening febrile illness. It is spread only through person to person or via oral-fecal route or through asymptomatic human carrier.
• Non typhoidal serotypes
It is worldwide disease of human and animals. It infects both animals and human. It causes gastroenteritis which is inflammation of GI tract leading to diarrhea, vomiting abdominal cramps which last for up to 7 days. It is also called as Salmonellosis. It is self-limiting and uncomplicated one. It not required hospitalizations and antibiotic treatment.

AFTER THE EXPOSURE TO ORGANISM, AT WHAT TIME THE SYMPTOMS OF TYPHOID FEVER APPEARED

Its take about 7-14 days (incubation period) or one-three weeks for the symptoms of typhoid fever to be appeared after exposure to disease. Larger infectious dose will reduce incubation period.

FACTORS FOR TYPHOID FEVER

1. Less hand washing habit particularly before taking meal and after defecation. (Transmission of disease occur through Oro-fecal route because ill person and carrier person of infections shed typhoid bacteria into the faces. If a person without washing hand handle the food, then that contaminated food become responsible for spread of typhoid infection).
2. Water contaminated with sewage for drinking and irrigation purposes
3. Poor sanitization
4. Contaminated food or under cook food
5. Use of ice or ice ice cubes made from contaminated water

WHAT IS MDR TYPHOID (MULTI DRUG RESISTANT) TYPHOID

MDR Typhoid is resistant form of typhoid fever especially to first line therapy which includes ampicillin, cotrimoxazole and chloramphenicol with or without resistance to second line of therapy. This resistant form of Salmonella Typhi first appeared in 1960.

WHY SALMONELLA TYPHI SHOW RESISTANCE IN MDR TYPHOID?

H58 clade containing gene in resistant isolates is responsible for resistance to first line therapy against typhoid fever.

IN 1990, CIPROFLOXACIN RESISTANCE AGAINST TYPHOID FEVER HAS ALSO BEEN REPORTED IN SOUTH ASIA. RECENTLY, AN EXTENSIVELY RESISTANT FORM OF TYPHOID FEVER IS BEING REPORTED FROM SOUTH ASIA. WHY RESISTANCE OF CIPROFLOXACIN IN SALMONELLA TYPHI HAS BEEN OBSERVED IN SOUTH ASIA?
The AMR in Salmonella typhi has been observed in South Asia because of self-medication. If people have diarrhea or gastroenteritis or uncomplicated salmonellosis then they take metronidazole first and if there is no improvement of symptoms then they take simply 2-3 doses of ciprofloxacin which improves symptoms. Secondly for positive and negative coverage physician prescribed ciprofloxacin without any evaluation and rationalization. The misuse or over use of broad-spectrum antibiotic is also one of the main causative factors of AMR in Salmonella Typhi.

WHAT IS XDR TYPHOID (EXTENSIVELY RESISTANT TYPHOID)

A first extensively resistant form of typhoid fever is first reported in Bangladesh and India. Afterwards, a largest numbers of blood culture contaminated XDR-TYPHOID FEVER are reported in Pakistan. XDR typhoid shows resistance to all recommended antibiotic therapy including ampicillin, cotrimoxazole, chloramphenicol, third generation cephalosporin, fluroquinolones. However, Pakistani XDR cases of typhoid fever are susceptible to only one oral antibiotic which is AZITHROMYCIN and IV EXTENDED SPECTRUM BETA LACTAM CARBAPENEM and TIGECYCLINE (parental) which restricting treatment options for physicians on one side while on other hand increases the threat to patients who might develop severe illness and parental treatment also increases the cost of treatment due to hospitalizations.

WHY SALMONELLA TYPHI IS SHOWING RESISTANCE TO ANTIBIOTICS

The reason is that they harbored a plasmid which carries a gene (blaCTX- M15) which is responsible for resistance to ceftriaxone and qnrs gene is responsible for resistance to fluroquinolones.

REASON OF AMR (ANTI MICROBIAL RESISTANCE) IN SALMONELLA TYPHI IN PAKISTAN

In recent decades, AMR has been increasingly prevalent in Salmonella Enterica Serovars Typhi. Anti-microbial therapy is the main treatment for typhoid fever. These successive waves of AMR in Salmonella Typhi left no choice of anti-microbial treatment in future in particular of oral anti-microbial therapy. XDR is a form of antimicrobial resistance (AMR). The following factors are mainly responsible for XDR anti-microbial resistance.

CONTRIBUTION OF TYPHI DOT TEST IN EMERGENCE OF AMR

Before to understand how the Typhi dot test is increasing AMR in third world country like Pakistan, we will try to understand how Typhi dot test performed.

TYPHI DOT TEST

Typhi dot test is performed on dot ELISA KIT against the outer membrane protein of SALMONELLA TYPHI which detects IgM and IgG antibodies. It is a qualitative test. After the 2-3 days of infection, the test is supposed to be positive and it is commonly used in endemic areas as a rapid serological diagnostic test. IgM antibodies show recent typhoid infection (early phase of infection) because when any antigen virus or bacteria attack the body IgM acts as first line defensive army of immune system while IgG antibodies indicate remote typhoid infection (past or chronic infection). Typhi dot test is supposed to be a easy, rapid reliable, affordable diagnostic test having promising results but in fact its low sensitivity and specificity causing misdiagnosis, wrong treatment and responsible for emergence of AMR.

INTERPRETATION OF TYPHI DOT TEST

• If test results show positive IgM antibodies, then it indicates early phase of infection.
• If test show positive IgM and IgG antibodies then it indicates middle phase of infection.
• If test results show only specific IgG antibodies, then it indicates chronic or past infection.

HOW THE WRONG INTERPRETATION OF TYPHOID DOT TEST LEAD TO MISDIAGNOSIS OF TYPHOID FEVER

The rise of only positive IgG antibodies is sometime responsible for wrong interpretation of typhoid fever. After recovering from typhoid illness, the IgG antibodies can prevail from more 2 years. Since it supposed to indicates past or current chronic infections so, it is unable to differentiates between current or past disease. In case of re-infection, there will be more pronounced effect of IgG due to secondary immune response over IgM that will hide the effects of IgM that will lead to misinterpretation of test results. This masking of IgM may lead to only IgG antibodies positive which may be ignored by considering test result is of past infection. In some cases, previous infection shows false positive result. Sometimes only IgG positive antibodies can be interpreted as state of current chronic infection (which is in fact due to previous preexisting immunity) may lead to misdiagnosis and commencing of antibiotics therapy lead to AMR.

PRE-EXISTING IMMUNITY

Due to past infection or immunization, there is pre-existing immunity especially with IgG antibodies which can result into false positive typhoid test.

ACCORDING TO PAKISTAN JOURNAL OF MEDICAL SCIENCE

According to Pakistan journal of medical science, Typhi dot test results versus blood culture results of patient with acute febrile illness of 3 or more days were analyzed at CIVIL HOSPITAL KARACHI in which it is concluded that the result of typhi dot test is not as promising as clinicians expect it to because of its low sensitivity and specificity. Following drawback are observed in this study which are as follow,
LOW SENSITIVITY, LOW SPECIFITY AND CROSS REACTIVITY

The O and H antigen of Salmonella Typhi are cross reactive with antibodies from other Salmonella serotypes and related gram-negative bacteria resulting in high positive false results. Non typhoidal Salmonella is also showing false positive results for typhoid fever. The false positive result of typhoid fever through Typhi dot test are also reporting in the other viral or bacterial disease like urinary tract infection, liver abscess, malaria, respiratory tract infection, pulmonary tuberculosis, dengue fever infection, gastroenteritis, tonsilitis, sinusitis, systemic lupus erythematosus and viral arthritis.

CROSS REACTIVITY OF S.TYPHI IgM IN DENGUE INFECTION VIRUS PATIENTS

During the outbreaks of dengue infection virus in Pakistan, a number of patients have positive Salmonella Typhi IgM results despite negative Salmonella Typhi cultures. This clear indicate that immunoassay is unable to differentiates between Salmonella Typhi infection and other infectious diseases and false positive result leading to erroneous management of patients. Therefore, commencing of antibiotics on the basis of false positive result leading to antimicrobial resistance (AMR) in large ERRONEOUS MANAGEMENT DUE TO LACK OF DIFFERENTIAL DIAGNOSIS (accurate diagnosis) THROUGH IMMUNOASSAY In endemic areas, because of nonspecific and common symptoms of typhoid fever with other viral or bacterial febrile illness, if patient is presented with persistent fever for more than 3 days then on the clinical basis it is misdiagnosed for typhoid fever. Immunoassay (widely test or Typhi dot test) are unable to differentiates among salmonellosis(gastroenteritis), others viral or bacterial illness and Typhoid fever So, misdiagnosis is leading to antibiotics resistant Salmonella Typhi in XDR form Accurate diagnosis is very important for fruitful and erroneous free management of patients. Diagnosis on clinical basis or on the basis of immunoassay false positive results is the key factor of erroneous management and prevalence of AMR.

HOW BROAD-SPECTRUM ANTIBIOTIC INCREASE THE RISK OF TYPHOID FEVER

Normal gastric acid PH and normal intestinal motility are first line defense against the low dose food borne infectious organism. Normal flora of gut also protective against these infectious organisms. Malnutrition and use of broad-spectrum antibiotic reduces the gut flora probably by altering anerobic intestinal flora and amplify risk of invasion of Salmonellae and incidence of typhoid fever.

STEP THAT PAKISTAN TAKEN TO STOP SPREAD OF XDR The following steps are taken by Pakistan to stop spread of XDR NATIONAL INSTITUTE OF HEALTH (NIH) OF PAKISTAN The NATIONAL INSTITUTE OF HEALTH OF PAKISTAN has directed the practitioners and health care facilities not to prescribed WIDAL OR TYPHI DOT TEST for confirmation of typhoid fever due to false positive results. Only stick to blood culture test for typhoid fever diagnosis. Because both tests are responsible for spread of AMR. Almost a decade ago, Widal test is discontinued across world and Typhi dot test is either stopped or discouraged in the most parts of world. Prescribing these existing out dated diagnostic methods causes the surge of XDR-TYPHOID.

MASS VACCINATION

There are 3 types of vaccine available for typhoid fever.
1. live attenuated oral vaccine
2. inactivated (killed) vaccine
3. Typhoid conjugate vaccine (TCVs)
Pakistan became the first country who introduced TCVs in routine immunization programs. All the children aged from 9 months to 15 years should get 0.5ml of TCVs vaccine into muscles irrespective of previously vaccinated or not.

CONCLUSION

Increasing cases of XDR typhoid fever in South Asia highlights the need of continuous genomic surveillance of Salmonella Typhi and there must be existence of strong reporting network through which private clinic, private medical centers, private practitioner and private hospitals can report these causes from the communities to concern higher health authorities in Pakistan. Although vaccination provide short term to intermediate control over the XDR outbreaks by decreasing need of antibiotics. However, Azithromycin should be reserve only for XDR-TYPHOID cases but during Corona pandemic mostly physician prescribed Azithromycin. Azithromycin was widely prescribed antibiotic. Unfortunately, private sector of health care departments in Pakistan is still prescribing Azithromycin without any investigation. Strict laws should be set in to discourage over-prescribing of antibiotic. Ultimate solution to stop this resistant outbreak of XDR-TYPHOID is availability of safe drink water (If safe drink water is not available then boil it), improve sanitization, mass vaccination, limit eat out from street vendors and avoid consumption of ice or ice cubes made from contaminated water. Irrational prescribing of antibiotics should be discouraged for antibiotic Stewardship. Immunoassay (widal and Typhi dot test) should be discouraged for typhoid fever confirmation which will be helpful in preventing AMR by making accurate and differential diagnosis. Only blood culture test should be used as diagnostic tool for typhoid fever.